Methods and compositions for treating pulmonary hypertension and related diseases and disorders

ABSTRACT

Methods of treating pulmonary hypertension are disclosed. Particular methods comprise the administration of a tryptophan hydroxylase inhibitor and at least one other active pharmaceutical ingredient to patient in need thereof. Pharmaceutical formulations are also disclosed.

This application claims priority to U.S. provisional application No.60/949,040, filed Jul. 11, 2007, the entirety of which is incorporatedherein by reference.

1. FIELD OF THE INVENTION

This invention relates to methods and compositions for the treatment ofpulmonary hypertension and related diseases and disorders.

2. BACKGROUND

Pulmonary hypertension (PH), or pulmonary arterial hypertension (PAH),is a disease characterized by increased pulmonary artery pressure andpulmonary vascular resistance. Harrison's Principles of InternalMedicine, 15^(th) ed., pp. 1506-1507 (McGraw-Hill, 2001). Leftuntreated, PH “usually has a dismal prognosis culminating in rightventricular failure and death.” Ulrich, S., et al., Swiss Med. Wkly137:73-82, 73 (2007).

In 2003, the World Health Organization (WHO) sponsored the developmentof guidelines, called the “Venice classification,” which are now used toclassify types of PH.http://www.tracleer.com/default.asp?page=CouldHave_WHO (accessed Jun.29, 2007). The first type, WHO Group 1.1, is idiopathic pulmonaryarterial hypertension (IPAH). This refers to PAH that occurs at random,without an apparent cause. IPAH used to be called “primary pulmonaryhypertension” or PPH. Id.

The second type, WHO Group 1.2, is familial pulmonary arterialhypertension (FPAH). With this type of PAH, a faulty gene is passed onthrough the family, which causes the PAH to develop over time. It isestimated that at least 6 to 10 percent of PAH cases occur in familieswhere at least one other person has had the disease. Id.

The third type, WHO Group 1.3, is pulmonary arterial hypertensionassociated with other diseases or conditions (APAH). This used to becalled “PAH secondary to other conditions” or Secondary PAH. Thiscategory includes PAH associated with collagen vascular disease or“connective tissue disease” (e.g., scleroderma (SSc)-including CRESTsyndrome-lupus (SLE)), congenital systemic-to-pulmonary shunts(congenital heart disease), portal hypertension, HIV infection, drugsand toxins, and other diseases and disorders (e.g., thyroid disorders,glycogen storage diseases, Gaucher disease, hereditary hemorrhagictelangiectasia, hemoglobinopathies, myeloproliferative disorders,splenectomy). Id.

The fourth type, WHO Group 1.4, is pulmonary arterial hypertensionassociated with significant venous or capillary involvement, andincludes pulmonary veno-occlusive disease (PVOD) and pulmonary capillaryhemangiomatosis (PCH).

The fifth and final type, WHO Group 1.5, is persistent pulmonaryhypertension of the newborn. Id.

Drugs currently used to treat PH include pulmonary vasodilators, calciumchannel blockers, and inhibitors of platelet aggregation. The MerckManual, 17^(th) ed., pp. 1703-4 (Merck Research Laboratories, 1999).Diuretics, nitric oxide, phosphodiesterase 5 inhibitors (e.g.,sildenafil) and endothelin receptor antagonists (ERAs) are also used forits treatment. Ulrich, S., et al., Swiss Med. Wkly 137:73-82, 76-77(2007). Endothelin receptor antagonists work by binding to the ET_(A)and/or ET_(B) receptor sites in the endothelium and vasculature smoothmuscle, thereby preventing the neurohormone endothelin-1 (ET-1) frombinding to these same receptor sites and triggering vasoconstriction.Id. at 76-77. An example of an ERA is bosentan (TRACLEER®).

Other methods of treating PH have been investigated. For example,selective serotonin reuptake inhibitors (SSRIs) reportedly reverse PH inrats. Id. at 79. These compounds, which are widely used to treatdepression, affect the reuptake of the neurotransmitter serotonin(5-HT).

Serotonin is synthesized in two steps from the amino acid tryptophan.Goodman & Gilman's The Pharmacological Basis of Therapeutics, 10^(th)ed., p. 270 (McGraw-Hill, 2001). The first step is rate-limiting, and iscatalyzed by the enzyme tryptophan hydroxylase (TPH), which has twoknown isoforms: TPH1, which is expressed in the periphery, and TPH2,which is expressed primarily in the brain. Walther, D. J., et al.,Science 299:76 (2003). Mice genetically deficient for the tph1 gene(“knockout mice”) have been reported. In one case, the mice reportedlyexpressed normal amounts of serotonin in classical serotonergic brainregions, but largely lacked serotonin in the periphery. Id. In another,the knockout mice exhibited abnormal cardiac activity, which wasattributed to a lack of peripheral serotonin. Côté, F., et al., PNAS100(23):13525-13530 (2003). Recently, TPH knockout mice were studied ina hypoxia-induced pulmonary arterial hypertension model. Morecroft, I.,et al., Hypertension 49:232-236 (2007). The results of those studiessuggest that TPH1 and peripheral serotonin “play an essential role inthe development of hypoxia-induced elevations in pulmonary pressures andhypoxia-induced pulmonary vascular remodeling.” Id. at 232.

3. SUMMARY OF THE INVENTION

This invention is directed, in part, to methods of treating pulmonaryhypertension and related diseases and disorders, which compriseadministering to a patient therapeutically effective amounts of atryptophan hydroxylase (TPH) inhibitor and at least one other activepharmaceutical ingredient.

One embodiment encompasses a method of treating, managing or preventingpulmonary hypertension, which comprises administering to a patient inneed thereof therapeutically or prophylactically effective amounts of anendothelin receptor antagonist and a tryptophan hydroxylase inhibitor.

Another encompasses a method of treating, managing or preventingpulmonary hypertension, which comprises administering to a patient inneed thereof therapeutically or prophylactically effective amounts of ananticoagulant and a tryptophan hydroxylase inhibitor.

Another encompasses a method of treating, managing or preventingpulmonary hypertension, which comprises administering to a patient inneed thereof therapeutically or prophylactically effective amounts of acalcium channel blocker and a tryptophan hydroxylase inhibitor.

Another encompasses a method of treating, managing or preventingpulmonary hypertension, which comprises administering to a patient inneed thereof therapeutically or prophylactically effective amounts of aprostacyclin and a tryptophan hydroxylase inhibitor.

Another encompasses a method of treating, managing or preventingpulmonary hypertension, which comprises administering to a patient inneed thereof therapeutically or prophylactically effective amounts ofnitric oxide or a nitric oxide precursor or releasing compound, and atryptophan hydroxylase inhibitor.

Another encompasses a method of treating, managing or preventingpulmonary hypertension, which comprises administering to a patient inneed thereof therapeutically or prophylactically effective amounts of aphosphodiesterase 5 inhibitor and a tryptophan hydroxylase inhibitor.

Another encompasses a method of treating, managing or preventingpulmonary hypertension, which comprises administering to a patient inneed thereof therapeutically or prophylactically effective amounts of adiuretic and a tryptophan hydroxylase inhibitor.

Another encompasses a method of treating, managing or preventingpulmonary hypertension, which comprises administering to a patient inneed thereof therapeutically or prophylactically effective amounts of aplatelet derived growth factor and a tryptophan hydroxylase inhibitor.

The invention also encompasses pharmaceutical formulations (e.g., singleunit dosage forms) comprising a TPH inhibitor and at least one otheractive pharmaceutical ingredient.

4. BRIEF DESCRIPTION OF THE FIGURE

Aspects of the invention may be understood with reference to theattached FIGURE.

FIG. 1 shows the effects of a potent TPH1 inhibitor of the invention inthe mouse gastrointestinal tract and brain after oral administration.All data are presented as percentage of the mean of the control(vehicle-dosed) group. Error bars are S.E.M. N=5 per group. The symbolsare *, p<0.05 vs control group. For the brain data, p=0.5, one-wayANOVA.

5. DETAILED DESCRIPTION

This invention is based, in part, on studies of tph1 knockout mice andthe discovery of compounds that inhibit tryptophan hydroxylase (e.g.,TPH1).

5.1. Definitions

Unless otherwise indicated, the term “alkenyl” means a straight chain,branched and/or cyclic hydrocarbon having from 2 to 20 (e.g., 2 to 10 or2 to 6) carbon atoms, and including at least one carbon-carbon doublebond. Representative alkenyl moieties include vinyl, allyl, 1-butenyl,2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl,2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, 1-hexenyl, 2-hexenyl,3-hexenyl, 1-heptenyl, 2-heptenyl, 3-heptenyl, 1-octenyl, 2-octenyl,3-octenyl, 1-nonenyl, 2-nonenyl, 3-nonenyl, 1-decenyl, 2-decenyl and3-decenyl.

Unless otherwise indicated, the term “alkyl” means a straight chain,branched and/or cyclic (“cycloalkyl”) hydrocarbon having from 1 to 20(e.g., 1 to 10 or 1 to 4) carbon atoms. Alkyl moieties having from 1 to4 carbons are referred to as “lower alkyl.” Examples of alkyl groupsinclude methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl,pentyl, hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl,2,2,4-trimethylpentyl, nonyl, decyl, undecyl and dodecyl. Cycloalkylmoieties may be monocyclic or multicyclic, and examples includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and adamantyl.Additional examples of alkyl moieties have linear, branched and/orcyclic portions (e.g., 1-ethyl-4-methyl-cyclohexyl). The term “alkyl”includes saturated hydrocarbons as well as alkenyl and alkynyl moieties.

Unless otherwise indicated, the term “alkoxy” means an —O-alkyl group.Examples of alkoxy groups include —OCH₃, —OCH₂CH₃, —O(CH₂)₂CH₃,—O(CH₂)₃CH₃, —O(CH₂)₄CH₃, and —O(CH₂)₅CH₃.

Unless otherwise indicated, the term “alkylaryl” or “alkyl-aryl” meansan alkyl moiety bound to an aryl moiety.

Unless otherwise indicated, the term “alkylheteroaryl” or“alkyl-heteroaryl” means an alkyl moiety bound to a heteroaryl moiety.

Unless otherwise indicated, the term “alkylheterocycle” or“alkyl-heterocycle” means an alkyl moiety bound to a heterocycle moiety.

Unless otherwise indicated, the term “alkynyl” means a straight chain,branched or cyclic hydrocarbon having from 2 to 20 (e.g., 2 to 20 or 2to 6) carbon atoms, and including at least one carbon-carbon triplebond. Representative alkynyl moieties include acetylenyl, propynyl,1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1-butynyl,4-pentynyl, 1-hexynyl, 2-hexynyl, 5-hexynyl, 1-heptynyl, 2-heptynyl,6-heptynyl, 1-octynyl, 2-octynyl, 7-octynyl, 1-nonynyl, 2-nonynyl,8-nonynyl, 1-decynyl, 2-decynyl and 9-decynyl.

Unless otherwise indicated, the term “aryl” means an aromatic ring or anaromatic or partially aromatic ring system composed of carbon andhydrogen atoms. An aryl moiety may comprise multiple rings bound orfused together. Examples of aryl moieties include anthracenyl, azulenyl,biphenyl, fluorenyl, indan, indenyl, naphthyl, phenanthrenyl, phenyl,1,2,3,4-tetrahydro-naphthalene, and tolyl.

Unless otherwise indicated, the term “arylalkyl” or “aryl-alkyl” meansan aryl moiety bound to an alkyl moiety.

Unless otherwise indicated, the terms “biohydrolyzable amide,”“biohydrolyzable ester,” “biohydrolyzable carbamate,” “biohydrolyzablecarbonate,” “biohydrolyzable ureido” and “biohydrolyzable phosphate”mean an amide, ester, carbamate, carbonate, ureido, or phosphate,respectively, of a compound that either: 1) does not interfere with thebiological activity of the compound but can confer upon that compoundadvantageous properties in vivo, such as uptake, duration of action, oronset of action; or 2) is biologically inactive but is converted in vivoto the biologically active compound. Examples of biohydrolyzable estersinclude lower alkyl esters, alkoxyacyloxy esters, alkyl acylamino alkylesters, and choline esters. Examples of biohydrolyzable amides includelower alkyl amides, α-amino acid amides, alkoxyacyl amides, andalkylaminoalkyl-carbonyl amides. Examples of biohydrolyzable carbamatesinclude lower alkylamines, substituted ethylenediamines, aminoacids,hydroxyalkylamines, heterocyclic and heteroaromatic amines, andpolyether amines.

Unless otherwise indicated, the terms “halogen” and “halo” encompassfluorine, chlorine, bromine, and iodine.

Unless otherwise indicated, the term “heteroalkyl” refers to an alkylmoiety (e.g., linear, branched or cyclic) in which at least one of itscarbon atoms has been replaced with a heteroatom (e.g., N, O or S).

Unless otherwise indicated, the term “heteroaryl” means an aryl moietywherein at least one of its carbon atoms has been replaced with aheteroatom (e.g., N, O or S). Examples include acridinyl,benzimidazolyl, benzofuranyl, benzoisothiazolyl, benzoisoxazolyl,benzoquinazolinyl, benzothiazolyl, benzoxazolyl, furyl, imidazolyl,indolyl, isothiazolyl, isoxazolyl, oxadiazolyl, oxazolyl, phthalazinyl,pyrazinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrimidyl,pyrrolyl, quinazolinyl, quinolinyl, tetrazolyl, thiazolyl, andtriazinyl.

Unless otherwise indicated, the term “heteroarylalkyl” or“heteroaryl-alkyl” means a heteroaryl moiety bound to an alkyl moiety.

Unless otherwise indicated, the term “heterocycle” refers to anaromatic, partially aromatic or non-aromatic monocyclic or polycyclicring or ring system comprised of carbon, hydrogen and at least oneheteroatom (e.g., N, O or S). A heterocycle may comprise multiple (i.e.,two or more) rings fused or bound together. Heterocycles includeheteroaryls. Examples include benzo[1,3]dioxolyl,2,3-dihydro-benzo[1,4]dioxinyl, cinnolinyl, furanyl, hydantoinyl,morpholinyl, oxetanyl, oxiranyl, piperazinyl, piperidinyl,pyrrolidinonyl, pyrrolidinyl, tetrahydrofuranyl, tetrahydropyranyl,tetrahydropyridinyl, tetrahydropyrimidinyl, tetrahydrothiophenyl,tetrahydrothiopyranyl and valerolactamyl.

Unless otherwise indicated, the term “heterocyclealkyl” or“heterocycle-alkyl” refers to a heterocycle moiety bound to an alkylmoiety.

Unless otherwise indicated, the term “heterocycloalkyl” refers to anon-aromatic heterocycle.

Unless otherwise indicated, the term “heterocycloalkylalkyl” or“heterocycloalkylalkyl” refers to a heterocycloalkyl moiety bound to analkyl moiety.

Unless otherwise indicated, the term “pharmaceutically acceptable salts”refers to salts prepared from pharmaceutically acceptable non-toxicacids or bases including inorganic acids and bases and organic acids andbases. Suitable pharmaceutically acceptable base addition salts includemetallic salts made from aluminum, calcium, lithium, magnesium,potassium, sodium and zinc or organic salts made from lysine,N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine,ethylenediamine, meglumine (N-methylglucamine) and procaine. Suitablenon-toxic acids include inorganic and organic acids such as acetic,alginic, anthranilic, benzenesulfonic, benzoic, camphorsulfonic, citric,ethenesulfonic, formic, fumaric, furoic, galacturonic, gluconic,glucuronic, glutamic, glycolic, hydrobromic, hydrochloric, isethionic,lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic,pantothenic, phenylacetic, phosphoric, propionic, salicylic, stearic,succinic, sulfanilic, sulfuric, tartaric acid, and p-toluenesulfonicacid. Specific non-toxic acids include hydrochloric, hydrobromic,phosphoric, sulfuric, and methanesulfonic acids. Examples of specificsalts thus include hydrochloride and mesylate salts. Others arewell-known in the art. See, e.g., Remington's Pharmaceutical Sciences,18^(th) ed. (Mack Publishing, Easton Pa.: 1990) and Remington: TheScience and Practice of Pharmacy, 19^(th) ed. (Mack Publishing, EastonPa.: 1995).

Unless otherwise indicated, the term “potent TPH1 inhibitor” is acompound that has a TPH1_IC₅₀ of less than about 10 μM.

Unless otherwise indicated, the term “prodrug” encompassespharmaceutically acceptable esters, carbonates, thiocarbonates, N-acylderivatives, N-acyloxyalkyl derivatives, quaternary derivatives oftertiary amines, N-Mannich bases, Schiff bases, amino acid conjugates,phosphate esters, metal salts and sulfonate esters of compoundsdisclosed herein. Examples of prodrugs include compounds that comprise abiohydrolyzable moiety (e.g., a biohydrolyzable amide, biohydrolyzablecarbamate, biohydrolyzable carbonate, biohydrolyzable ester,biohydrolyzable phosphate, or biohydrolyzable ureide analog). Prodrugsof compounds disclosed herein are readily envisioned and prepared bythose of ordinary skill in the art. See, e.g., Design of Prodrugs,Bundgaard, A. Ed., Elseview, 1985; Bundgaard, H., “Design andApplication of Prodrugs,” A Textbook of Drug Design and Development,Krosgaard-Larsen and H. Bundgaard, Ed., 1991, Chapter 5, p. 113-191; andBundgaard, H., Advanced Drug Delivery Review, 1992, 8, 1-38.

Unless otherwise indicated, a “prophylactically effective amount” of acompound is an amount sufficient to prevent a disease or condition, orone or more symptoms associated with the disease or condition, orprevent its recurrence. A prophylactically effective amount of acompound is an amount of therapeutic agent, alone or in combination withother agents, which provides a prophylactic benefit in the prevention ofthe disease. The term “prophylactically effective amount” can encompassan amount that improves overall prophylaxis or enhances the prophylacticefficacy of another prophylactic agent.

Unless otherwise indicated, the term “protecting group” or “protectivegroup,” when used to refer to part of a molecule subjected to a chemicalreaction, means a chemical moiety that is not reactive under theconditions of that chemical reaction, and which may be removed toprovide a moiety that is reactive under those conditions. Protectinggroups are well known in the art. See, e.g., Greene, T. W. and Wuts, P.G. M., Protective Groups in Organic Synthesis (3^(rd) ed., John Wiley &Sons: 1999); Larock, R. C., Comprehensive Organic Transformations(2^(nd) ed., John Wiley & Sons: 1999). Some examples include benzyl,diphenylmethyl, trityl, Cbz, Boc, Fmoc, methoxycarbonyl, ethoxycarbonyl,and pthalimido.

Unless otherwise indicated, the term “pseudohalogen” refers to apolyatomic anion that resembles a halide ion in its acid-base,substitution, and redox chemistry, generally has low basicity, and formsa free radical under atom transfer radical polymerization conditions.Examples of pseudohalogens include azide ions, cyanide, cyanate,thiocyanate, thiosulfate, sulfonates, and sulfonyl halides.

Unless otherwise indicated, the term “selective TPH1 inhibitor” is acompound that has a TPH2_IC₅₀ that is at least about 10 times greaterthan its TPH1_IC₅₀.

Unless otherwise indicated, the term “serotonin-mediated adverse effect”refers to an adverse effect that is attributable to increased levels ofperipheral 5-hydroxytryptamine (5-HT).

Unless otherwise indicated, the term “stereomerically enrichedcomposition of” a compound refers to a mixture of the named compound andits stereoisomer(s) that contains more of the named compound than itsstereoisomer(s). For example, a stereoisomerically enriched compositionof (S)-butan-2-ol encompasses mixtures of (S)-butan-2-ol and(R)-butan-2-ol in ratios of, e.g., about 60/40, 70/30, 80/20, 90/10,95/5, and 98/2.

Unless otherwise indicated, the term “stereoisomeric mixture”encompasses racemic mixtures as well as stereomerically enrichedmixtures (e.g., R/S=30/70, 35/65, 40/60, 45/55, 55/45, 60/40, 65/35 and70/30).

Unless otherwise indicated, the term “stereomerically pure” means acomposition that comprises one stereoisomer of a compound and issubstantially free of other stereoisomers of that compound. For example,a stereomerically pure composition of a compound having one stereocenterwill be substantially free of the opposite stereoisomer of the compound.A stereomerically pure composition of a compound having twostereocenters will be substantially free of other diastereomers of thecompound. A typical stereomerically pure compound comprises greater thanabout 80% by weight of one stereoisomer of the compound and less thanabout 20% by weight of other stereoisomers of the compound, greater thanabout 90% by weight of one stereoisomer of the compound and less thanabout 10% by weight of the other stereoisomers of the compound, greaterthan about 95% by weight of one stereoisomer of the compound and lessthan about 5% by weight of the other stereoisomers of the compound,greater than about 97% by weight of one stereoisomer of the compound andless than about 3% by weight of the other stereoisomers of the compound,or greater than about 99% by weight of one stereoisomer of the compoundand less than about 1% by weight of the other stereoisomers of thecompound.

Unless otherwise indicated, the term “substituted,” when used todescribe a chemical structure or moiety, refers to a derivative of thatstructure or moiety wherein one or more of its hydrogen atoms issubstituted with an atom, chemical moiety or functional group such as,but not limited to, alcohol, aldehylde, alkoxy, alkanoyloxy,alkoxycarbonyl, alkenyl, alkyl (e.g., methyl, ethyl, propyl, t-butyl),alkynyl, alkylcarbonyloxy (—OC(O)alkyl), amide (—C(O)NH-alkyl- or-alkylNHC(O)alkyl), amidinyl (—C(NH)NH-alkyl or —C(NR)NH₂), amine(primary, secondary and tertiary such as alkylamino, arylamino,arylalkylamino), aroyl, aryl, aryloxy, azo, carbamoyl (—NHC(O)O-alkyl-or —OC(O)NH-alkyl), carbamyl (e.g., CONH₂, as well as CONH-alkyl,CONH-aryl, and CONH-arylalkyl), carbonyl, carboxyl, carboxylic acid,carboxylic acid anhydride, carboxylic acid chloride, cyano, ester,epoxide, ether (e.g., methoxy, ethoxy), guanidino, halo, haloalkyl(e.g., —CCl₃, —CF₃, —C(CF₃)₃), heteroalkyl, hemiacetal, imine (primaryand secondary), isocyanate, isothiocyanate, ketone, nitrile, nitro,oxygen (i.e., to provide an oxo group), phosphodiester, sulfide,sulfonamido (e.g., SO₂NH₂), sulfone, sulfonyl (including alkylsulfonyl,arylsulfonyl and arylalkylsulfonyl), sulfoxide, thiol (e.g., sulfhydryl,thioether) and urea (—NHCONH-alkyl-).

Unless otherwise indicated, a “therapeutically effective amount” of acompound is an amount sufficient to provide a therapeutic benefit in thetreatment or management of a disease or condition, or to delay orminimize one or more symptoms associated with the disease or condition.A therapeutically effective amount of a compound is an amount oftherapeutic agent, alone or in combination with other therapies, whichprovides a therapeutic benefit in the treatment or management of thedisease or condition. The term “therapeutically effective amount” canencompass an amount that improves overall therapy, reduces or avoidssymptoms or causes of a disease or condition, or enhances thetherapeutic efficacy of another therapeutic agent.

Unless otherwise indicated, the term “TPH1_IC₅₀” is the IC₅₀ of acompound for TPH1 as determined using the in vitro inhibition assaydescribed in the Examples, below.

Unless otherwise indicated, the term “TPH2_IC₅₀” is the IC₅₀ of acompound for TPH2 as determined using the in vitro inhibition assaydescribed in the Examples, below.

Unless otherwise indicated, the terms “treat,” “treating” and“treatment” contemplate an action that occurs while a patient issuffering from the specified disease or disorder, which reduces theseverity of the disease or disorder, or one or more of its symptoms, orretards or slows the progression of the disease or disorder.

Unless otherwise indicated, the term “include” has the same meaning as“include” and the term “includes” has the same meaning as “includes, butis not limited to.” Similarly, the term “such as” has the same meaningas the term “such as, but not limited to.”

Unless otherwise indicated, one or more adjectives immediately precedinga series of nouns is to be construed as applying to each of the nouns.For example, the phrase “optionally substituted alky, aryl, orheteroaryl” has the same meaning as “optionally substituted alky,optionally substituted aryl, or optionally substituted heteroaryl.”

It should be noted that a chemical moiety that forms part of a largercompound may be described herein using a name commonly accorded it whenit exists as a single molecule or a name commonly accorded its radical.For example, the terms “pyridine” and “pyridyl” are accorded the samemeaning when used to describe a moiety attached to other chemicalmoieties. Thus, the two phrases “XOH, wherein X is pyridyl” and “XOH,wherein X is pyridine” are accorded the same meaning, and encompass thecompounds pyridin-2-ol, pyridin-3-ol and pyridin-4-ol.

It should also be noted that if the stereochemistry of a structure or aportion of a structure is not indicated with, for example, bold ordashed lines, the structure or the portion of the structure is to beinterpreted as encompassing all stereoisomers of it. Similarly, names ofcompounds having one or more chiral centers that do not specify thestereochemistry of those centers encompass pure stereoisomers andmixtures thereof. Moreover, any atom shown in a drawing with unsatisfiedvalences is assumed to be attached to enough hydrogen atoms to satisfythe valences. In addition, chemical bonds depicted with one solid lineparallel to one dashed line encompass both single and double (e.g.,aromatic) bonds, if valences permit.

5.2. TPH Inhibitors

Particular embodiments of this invention utilize compounds capable ofinhibiting tryptophan hydroxylase (TPH). Preferred compounds are potentTPH1 inhibitors. Examples of potent TPH1 inhibitors are disclosed inU.S. patent application Ser. No. 11/638,677, filed Dec. 12, 2006.

Particular embodiments utilize compounds of formula I:

and pharmaceutically acceptable salts and solvates thereof, wherein: Ais optionally substituted cycloalkyl, aryl, or heterocycle; X is a bond,—O—, —S—, —C(O)—, —C(R₄)═, ═C(R₄)—, —C(R₃R₄)—, —C(R₄)═C(R₄)—, —C≡C—,—N(R₅)—, —N(R₅)C(O)N(R₅)—, —C(R₃R₄)N(R₅)—, —N(R₅)C(R₃R₄)—, —ONC(R₃)—,—C(R₃)NO—, —C(R₃R₄)O—, —OC(R₃R₄)—, —S(O₂)—, —S(O₂)N(R₅)—, —N(R₅)S(O₂)—,—C(R₃R₄)S(O₂)—, or —S(O₂)C(R₃R₄)—; D is optionally substituted aryl orheterocycle; R₁ is hydrogen or optionally substituted alkyl, alkyl-aryl,alkyl-heterocycle, aryl, or heterocycle; R₂ is hydrogen or optionallysubstituted alkyl, alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle;R₃ is hydrogen, alkoxy, amino, cyano, halogen, hydroxyl, or optionallysubstituted alkyl; R₄ is hydrogen, alkoxy, amino, cyano, halogen,hydroxyl, or optionally substituted alkyl or aryl; each R₅ isindependently hydrogen or optionally substituted alkyl or aryl; and n is0-3.

Particular compounds are of formula I(A):

Others are of formula II:

and pharmaceutically acceptable salts and solvates thereof, wherein: Ais optionally substituted cycloalkyl, aryl, or heterocycle; X is a bond,—O—, —S—, —C(O)—, —C(R₄)═, ═C(R₄)—, —C(R₃R₄)—, —C(R₄)═C(R₄)—, —C≡C—,—N(R₅)—, —N(R₅)C(O)N(R₅)—, —C(R₃R₄)N(R₅)—, —N(R₅)C(R₃R₄)—, —ONC(R₃)—,—C(R₃)NO—, —C(R₃R₄)O—, —OC(R₃R₄)—, —S(O₂)—, —S(O₂)N(R₅)—, —N(R₅)S(O₂)—,—C(R₃R₄)S(O₂)—, or —S(O₂)C(R₃R₄)—; D is optionally substituted aryl orheterocycle; E is optionally substituted aryl or heterocycle; R₁ ishydrogen or optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle,aryl, or heterocycle; R₂ is hydrogen or optionally substituted alkyl,alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle; R₃ is hydrogen,alkoxy, amino, cyano, halogen, hydroxyl, or optionally substitutedalkyl; R₄ is hydrogen, alkoxy, amino, cyano, halogen, hydroxyl, oroptionally substituted alkyl or aryl; R₅ is hydrogen or optionallysubstituted alkyl or aryl; and n is 0-3.

Particular compounds are of formula II(A):

With regard to the formulae disclosed herein (e.g., I, I(A), II andII(A)), particular compounds include those wherein A is optionallysubstituted cycloalkyl (e.g., 6-membered and 5-membered). In some, A isoptionally substituted aryl (e.g., phenyl or naphthyl). In others, A isoptionally substituted heterocycle (e.g., 6-membered and 5-membered).Examples of 6-membered heterocycles include pyridine, pyridazine,pyrimidine, pyrazine, and triazine. Examples of 5-membered heterocyclesinclude pyrrole, imidazole, triazole, thiazole, thiophene, and furan. Insome compounds, A is aromatic. In others, A is not aromatic. In some, Ais an optionally substituted bicyclic moiety (e.g., indole, iso-indole,pyrrolo-pyridine, or napthylene).

Particular compounds are of the formula:

wherein: each of A₁ and A₂ is independently a monocyclic optionallysubstituted cycloalkyl, aryl, or heterocycle. Compounds encompassed bythis formula include those wherein A₁ and/or A₂ is optionallysubstituted cycloalkyl (e.g., 6-membered and 5-membered). In some, A₁and/or A₂ is optionally substituted aryl (e.g., phenyl or naphthyl). Inothers, A₁ and/or A₂ is optionally substituted heterocycle (e.g.,6-membered and 5-membered). Examples of 6-membered heterocycles includepyridine, pyridazine, pyrimidine, pyrazine, and triazine. Examples of5-membered heterocycles include pyrrole, imidazole, triazole, thiazole,thiophene, and furan. In some compounds, A₁ and/or A₂ is aromatic. Inothers, A₁ and/or A₂ is not aromatic.

With regard to the formulae disclosed herein, particular compoundsinclude those wherein D is optionally substituted aryl (e.g., phenyl ornaphthyl). In others, D is optionally substituted heterocycle (e.g.,6-membered and 5-membered). Examples of 6-membered heterocycles includepyridine, pyridazine, pyrimidine, pyrazine, and triazine. Examples of5-membered heterocycles include pyrrole, imidazole, triazole, thiazole,thiophene, and furan. In some compounds, D is aromatic. In others, D isnot aromatic. In some, D is an optionally substituted bicyclic moiety(e.g., indole, iso-indole, pyrrolo-pyridine, or napthylene).

With regard to the various formulae disclosed herein, particularcompounds include those wherein E is optionally substituted aryl (e.g.,phenyl or naphthyl). In others, E is optionally substituted heterocycle(e.g., 6-membered and 5-membered). Examples of 6-membered heterocyclesinclude pyridine, pyridazine, pyrimidine, pyrazine, and triazine.Examples of 5-membered heterocycles include pyrrole, imidazole,triazole, thiazole, thiophene, and furan. In some compounds, E isaromatic. In others, E is not aromatic. In some, E is an optionallysubstituted bicyclic moiety (e.g., indole, iso-indole, pyrrolo-pyridine,or napthylene).

With regard to the various formulae disclosed herein, particularcompounds include those wherein R₁ is hydrogen or optionally substitutedalkyl.

In some, R₂ is hydrogen or optionally substituted alkyl.

In some, n is 1 or 2.

In some, X is a bond or S. In others, X is —C(R₄)═, ═C(R₄)—, —C(R₃R₄)—,—C(R₄)═C(R₄)—, or —C≡C—, and, for example, R₄ is independently hydrogenor optionally substituted alkyl. In others, X is —O—, —C(R₃R₄)O—, or—OC(R₃R₄)—, and, for example, R₃ is hydrogen or optionally substitutedalkyl, and R₄ is hydrogen or optionally substituted alkyl. In some, R₃is hydrogen and R₄ is trifluoromethyl. In some compounds, X is —S(O₂)—,—S(O₂)N(R₅)—, —N(R₅)S(O₂)—, —C(R₃R₄)S(O₂)—, or —S(O₂)C(R₃R₄)—, and, forexample, R₃ is hydrogen or optionally substituted alkyl, R₄ is hydrogenor optionally substituted alkyl, and R₅ is hydrogen or optionallysubstituted alkyl. In others, X is —N(R₅)—, —N(R₅)C(O)N(R₅)—,—C(R₃R₄)N(R₅)—, or —N(R₅)C(R₃R₄)—, and, for example, R₃ is hydrogen oroptionally substituted alkyl, R₄ is hydrogen or optionally substitutedalkyl, and each R₅ is independently hydrogen or optionally substitutedalkyl.

Other compounds are of the formula:

wherein, for example, R₃ is trifluoromethyl. Others are encompassed bythe formula:

wherein, for example, R₃ is hydrogen.

Some compounds are encompassed by the formula:

wherein: each of Z₁, Z₂, Z₃, and Z₄ is independently N or CR₆; each R₆is independently hydrogen, cyano, halogen, OR₇, NR₈R₉, amino, hydroxyl,or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; eachR₇ is independently hydrogen or optionally substituted alkyl, alkyl-arylor alkyl-heterocycle; each R₈ is independently hydrogen or optionallysubstituted alkyl, alkyl-aryl or alkyl-heterocycle; each R₉ isindependently hydrogen or optionally substituted alkyl, alkyl-aryl oralkyl-heterocycle; and m is 1-4. Certain such compounds are of theformula:

Others are of the formula:

wherein, for example, R₃ is trifluoromethyl. Others are of the formula:

wherein, for example, R₃ is hydrogen.

Referring to the various formulae above, some compounds are such thatall of Z₁, Z₂, Z₃, and Z₄ are N. In others, only three of Z₁, Z₂, Z₃,and Z₄ are N. In others, only two of Z₁, Z₂, Z₃, and Z₄ are N. Inothers, only one of Z₁, Z₂, Z₃, and Z₄ is N. In others, none of Z₁, Z₂,Z₃, and Z₄ are N.

Some compounds are of the formula:

wherein: each of Z′₁, Z′₂, and Z′₃ is independently N, NH, S, O or CR₆;each R₆ is independently amino, cyano, halogen, hydrogen, OR₇, SR₇,NR₈R₉, or optionally substituted alkyl, alkyl-aryl or alkyl-heterocycle;each R₇ is independently hydrogen or optionally substituted alkyl,alkyl-aryl or alkyl-heterocycle; each R₈ is independently hydrogen oroptionally substituted alkyl, alkyl-aryl or alkyl-heterocycle; each R₉is independently hydrogen or optionally substituted alkyl, alkyl-aryl oralkyl-heterocycle; and p is 1-3. Certain such compounds are of theformula:

Others are of the formula:

wherein, for example, R₃ is trifluoromethyl. Others are of the formula:

wherein, for example, R₃ is hydrogen.

Referring to the various formulae above, some compounds are such thatall of Z′₁, Z′₂, and Z′₃ are N or NH. In others, only two of Z′₁, Z′₂,and Z′₃ are N or NH. In others, only one of Z′₁, Z′₂, and Z′₃ is N orNH. In others, none of Z′₁, Z′₂, and Z′₃ are N or NH.

Some compounds are encompassed by the formula:

wherein: each of Z″₁, Z″₂, Z″₃, and Z″₄ is independently N or CR₁₀; eachR₁₀ is independently amino, cyano, halogen, hydrogen, OR₁₁, SR₁₁,NR₁₂R₁₃, or optionally substituted alkyl, alkyl-aryl oralkyl-heterocycle; each R₁₁ is independently hydrogen or optionallysubstituted alkyl, alkyl-aryl or alkyl-heterocycle; each R₁₂ isindependently hydrogen or optionally substituted alkyl, alkyl-aryl oralkyl-heterocycle; and each R₁₃ is independently hydrogen or optionallysubstituted alkyl, alkyl-aryl or alkyl-heterocycle. Certain suchcompounds are of the formula:

Others are of the formula:

wherein, for example, R₃ is trifluoromethyl. Others are of the formula:

wherein, for example, R₃ is hydrogen.

Referring to the various formulae above, some compounds are such thatall of Z″₁, Z″₂, Z″₃, and Z″₄ are N. In others, only three of Z″₁, Z″₂,Z″₃, and Z″₄ are N. In others, only two of Z″₁, Z″₂, Z″₃, and Z″₄ are N.In others, only one of Z″₁, Z″₂, Z″₃, and Z″₄ is N. In others, none ofZ″₁, Z″₂, Z″₃, and Z″₄ are N.

Some compounds are of the formula:

wherein: each of Z″₁, Z″₂, Z″₃, and Z″₄ is independently N or CR₁₀; eachR₁₀ is independently amino, cyano, halogen, hydrogen, OR₁₁, SR₁₁,NR₁₂R₁₃, or optionally substituted alkyl, alkyl-aryl oralkyl-heterocycle; each R₁₁ is independently hydrogen or optionallysubstituted alkyl, alkyl-aryl or alkyl-heterocycle; each R₁₂ isindependently hydrogen or optionally substituted alkyl, alkyl-aryl oralkyl-heterocycle; and each R₁₃ is independently hydrogen or optionallysubstituted alkyl, alkyl-aryl or alkyl-heterocycle. Certain suchcompounds are of the formula:

Others are of the formula:

wherein, for example, R₃ is trifluoromethyl. Others are of the formula:

wherein, for example, R₃ is hydrogen.

Referring to the various formulae above, some compounds are such thatall of Z″₁, Z″₂, Z″₃, and Z″₄ are N. In others, only three of Z″₁, Z″₂,Z″₃, and Z″₄ are N. In others, only two of Z″₁, Z″₂, Z″₃, and Z″₄ are N.In others, only one of Z″₁, Z″₂, Z″₃, and Z″₄ is N. In others, none ofZ″₁, Z″₂, Z″₃, and Z″₄ are N.

Some are of the formula:

the substituents of which are defined herein. Others are of the formula:

the substituents of which are defined herein. Others are of the formula:

the substituents of which are defined herein. Others are of the formula:

the substituents of which are defined herein.

Referring to the various formulae disclosed herein, particular compoundsinclude those wherein both A and E are optionally substituted phenyland, for example, X is —O—, —C(R₃R₄)O—, or —OC(R₃R₄)— and, for example,R₃ is hydrogen and R₄ is trifluoromethyl and, for example, n is 1.

This invention encompasses stereomerically pure compounds andstereomerically enriched compositions of them. Stereoisomers may beasymmetrically synthesized or resolved using standard techniques such aschiral columns, chiral resolving agents, or enzymatic resolution. See,e.g., Jacques, J., et al., Enantiomers, Racemates and Resolutions (WileyInterscience, New York, 1981); Wilen, S. H., et al., Tetrahedron 33:2725(1977); Eliel, E. L., Stereochemistry of Carbon Compounds (McGraw Hill,N.Y., 1962); and Wilen, S. H., Tables of Resolving Agents and OpticalResolutions, p. 268 (E. L. Eliel, Ed., Univ. of Notre Dame Press, NotreDame, Ind., 1972).

Particular compounds of the invention are potent TPH1 inhibitors.Specific compounds have a TPH1_IC₅₀ of less than about 10, 5, 2.5, 1,0.75, 0.5, 0.4, 0.3, 0.2, 0.1, or 0.05 μM.

Particular compounds are selective TPH1 inhibitors. Specific compoundshave a TPH1_IC₅₀ that is about 10, 25, 50, 100, 250, 500, or 1000 timesless than their TPH2_IC₅₀.

Particular compounds do not significantly inhibit human tyrosinehydroxylase (TH). For example, specific compounds have an IC₅₀ for TH ofgreater than about 100, 250, 500 or 1000 μm.

Particular compounds do not significantly inhibit human phenylalaninehydroxylase (PAH). For example, specific compounds have an IC₅₀ for PAHof greater than about 100, 250, 500 or 1000 μM.

Particular compounds of the invention do not significantly bind (e.g.,inhibit with an IC₅₀ of greater than about 10, 25, 50, 100, 250, 500,750, or 1000 μM) to one or more of the following: angiotensin convertingenzyme, erythropoietin (EPO) receptor, factor IX, factor XI, integrin(e.g., α4), isoxazoline or isoxazole fibrinogen receptor,metalloprotease, neutral endopeptidase (NEP), phosphatase (e.g.,tyrosine phosphatase), phosphodiesterase (e.g., PDE-4), polymerase,PPARγ, TNF-α, vascular cell adhesion molecule-1 (VCAM-1), or thevitronectin receptor. The ability of a compound to bind to (e.g.,inhibit) any of these targets can be readily determined using methodsknown in the art, as described in references cited above. Specificcompounds of the invention do not inhibit cell adhesion.

When administered to mammals (e.g., mice, rats, dogs, monkeys orhumans), certain compounds of the invention do not readily cross theblood/brain barrier (e.g., less than about 5, 2.5, 2, 1.5, 1, 0.5, or0.01 percent of compound in the blood passes into the brain). Theability or inability of a compound to cross the blood/brain barrier canbe determined by methods known in the art. See, e.g., Riant, P. et al.,Journal of Neurochemistry 51:421-425 (1988); Kastin, A. J., Akerstrom,V., J. Pharmacol. Exp. Therapeutics 294:633-636 (2000); W. A. Banks, W.A., et al., J. Pharmacol. Exp. Therapeutics 302:1062-1069 (2002).

5.3. Synthesis of TPH Inhibitors

Compounds of the invention can be prepared by methods known in the art,and by methods described herein.

For example, with reference to formula I, compounds in which E is phenyland D is optionally substituted pyrazine, pyridiazine, pyridine orphenyl can generally be prepared by the method shown in Scheme 1:

wherein, for example:

Compounds wherein X is —OCR₃— can generally be prepared using the methodshown in Scheme 2, wherein R₃ is CF₃ and D is pyrimidine:

wherein, for example, A is optionally substituted phenyl, biphenyl ornapthyl.

Compounds of the invention can also be prepared using the approach shownbelow in Scheme 3:

wherein P₁ is R₁ or a protecting group; P₂ is a protecting group; P₃ isOR₂ or a protecting group; X′ is, for example, O or N; Y₁ and Y₃ arehalogen (e.g., Br, Cl) or an appropriate pseudohalide (e.g., triflate);and each R′ is independently hydrogen or optionally substituted alkyl,alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle, or are takentogether with the oxygen atoms to which they are attached to provide acyclic dioxaborolane (e.g., 4,4,5,5-tetramethyl-1,3,2-dioxaborolane).The groups A, R₁, R₂, R₃, R₆ and m are defined elsewhere herein. Themoieties Z″₁, Z″₂, Z″₃, and Z″₄ are also defined herein, although it isto be understood that with regard to the scheme shown above, one of themis attached to the phenyl ring. For example, Z″₁ and Z″₄ may beindependently CR₁₀ (which is defined herein), while Z″₂ is N and Z″₃ isa carbon atom bound to the adjacent phenyl ring.

The individual reactions shown above can be performed using conditionsknown in the art. For example, palladium catalysts and conditionssuitable for the Suzuki coupling of the boron and halogen-containingmoieties are well known, and examples are provided below. In addition,types and appropriate uses of protecting groups are well known, as aremethods of their removal and replacement with moieties such as, but notlimited to, hydrogen (e.g., hydrolysis under acidic or basicconditions).

The A moiety can be bicyclic (e.g., optionally substituted biphenyl). Insuch cases, the starting material containing A can be prepared as shownbelow:

wherein Y₂ is halogen or pseudohalogen, and each R is independentlyhydrogen or optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle,aryl, or heterocycle, or are taken together with the oxygen atoms towhich they are attached to provide a cyclic dioxaborolane (e.g.,4,4,5,5-tetramethyl-1,3,2-dioxaborolane).

Another approach to the preparation of compounds wherein D is optionallysubstituted pyrimidine or triazine is shown below in Scheme 4:

wherein, for example, X is N, O or S, and FG is defined below:

-   -   FG=B(OH)₂ when E is optionally substituted Phenyl

-   -    when E is:

-   -   FG=H when E is:

Ester derivatives of these and other compounds of the invention can bereadily prepared using methods such as that shown below in Scheme 5,wherein E is optionally substituted phenyl:

An alternate approach to the preparation of triazine-based compounds isshown below in Scheme 6:

The cyclic moiety D can be any of a variety of structures, which arereadily incorporated into compounds of the invention. For example,compounds wherein D is oxazole can be prepared as shown below in Scheme7:

Using methods known in the art, the synthetic approaches shown above arereadily modified to obtain a wide range of compounds. For example,chiral chromatography and other techniques known in the art may be usedto separate stereoisomers of the final product. See, e.g., Jacques, J.,et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, NewYork, 1981); Wilen, S. H., et al., Tetrahedron 33:2725 (1977); Eliel, E.L., Stereochemistry of Carbon Compounds (McGraw Hill, N.Y., 1962); andWilen, S. H., Tables of Resolving Agents and Optical Resolutions, p. 268(E. L. Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind., 1972).In addition, as shown in some of the schemes above, syntheses mayutilize chiral starting materials to yield stereomerically enriched orpure products.

5.4. Methods of Use

This invention is directed, in part, to methods of treating pulmonaryhypertension and related diseases and disorders. Pulmonary hypertensionincludes idiopathic and familial PH. It also includes PH associated withother diseases or conditions (e.g., collagen vascular disease,congenital systemic-to-pulmonary shunts, a drug or toxin, Gaucherdisease, a glycogen storage disease, hereditary hemorrhagictelangiectasia, hemoglobinopathies, HIV infection, a myeloproliferativedisorder, portal hypertension, splenectomy, a thyroid disorder). It alsoincludes PAH and PAH associated with significant venous or capillaryinvolvement. It also includes PH associated with pulmonaryveno-occlusive disease or pulmonary capillary hemangiomatosis. It alsoincludes persistent PH of the newborn.

Particular methods comprise administering to a patient therapeuticallyor prophylactically effective amounts of a tryptophan hydroxylase (TPH)inhibitor and at least one second active pharmaceutical ingredient.Second active pharmaceutical ingredients include those that can affectvasodilation or vasoconstriction.

One embodiment of the invention encompasses a method of treating,managing or preventing pulmonary hypertension, which comprisesadministering to a patient in need thereof therapeutically orprophylactically effective amounts of an endothelin receptor antagonistand a tryptophan hydroxylase inhibitor. Examples of endothelin receptorantagonists include those which antagonize the ET_(A) receptor, theET_(B) receptor, or both the ET_(A) and ET_(B) receptors. Particularendothelin receptor antagonists include ambrisentan, BMS-193884,bosentan, darusentan, SB-234551, sitaxsentan, and tezosentan.

Another embodiment encompasses a method of treating, managing orpreventing pulmonary hypertension, which comprises administering to apatient in need thereof therapeutically or prophylactically effectiveamounts of an anticoagulant and a tryptophan hydroxylase inhibitor.Examples of anticoagulants include vitamin K antagonists (e.g.,warfarin, acenocoumarol, phenprocoumon, phenindione), herparin andderivatives thereof (e.g., fondaparinux), and direct thrombin inhibitors(e.g., argatroban, bivalirudin, lepirudin, ximelagatran).

Another embodiment encompasses a method of treating, managing orpreventing pulmonary hypertension, which comprises administering to apatient in need thereof therapeutically or prophylactically effectiveamounts of a calcium channel blocker and a tryptophan hydroxylaseinhibitor. Examples of calcium channel blockers include dihydropyridines(e.g., amlodipine, felodipine, nicardipine, nifedipine, nimodipine,nisoldipine, nitrenidipine, lacidipine, lercanidipine),phenylalkylamines (e.g., verapamil, gallopamil), benzothiazepines (e.g.,diltiazem), and menthol.

Another embodiment encompasses a method of treating, managing orpreventing pulmonary hypertension, which comprises administering to apatient in need thereof therapeutically or prophylactically effectiveamounts of a prostacyclin and a tryptophan hydroxylase inhibitor.Examples of prostacyclind include epoprostenol, iloprost andtreprostinil.

Another embodiment encompasses a method of treating, managing orpreventing pulmonary hypertension, which comprises administering to apatient in need thereof therapeutically or prophylactically effectiveamounts of nitric oxide or a nitric oxide precursor or releasingcompound, and a tryptophan hydroxylase inhibitor.

Another encompasses a method of treating, managing or preventingpulmonary hypertension, which comprises administering to a patient inneed thereof therapeutically or prophylactically effective amounts of aphosphodiesterase 5 inhibitor and a tryptophan hydroxylase inhibitor.Examples of phosphodiesterase 5 inhibitors include sildenafil,tadalafil, and vardenafil.

Another embodiment encompasses a method of treating, managing orpreventing pulmonary hypertension, which comprises administering to apatient in need thereof therapeutically or prophylactically effectiveamounts of a diuretic and a tryptophan hydroxylase inhibitor. Examplesof diuretics include high ceiling loop diuretics (e.g., furosemide,ethacrynic acid, torasemide, bumetanide), thiazides (e.g.,hydrochlorothiazide), potassium sparing diuretics (e.g.,spironolactone), and osmotic diuretics (e.g., mannitol).

Another embodiment encompasses a method of treating, managing orpreventing pulmonary hypertension, which comprises administering to apatient in need thereof therapeutically or prophylactically effectiveamounts of a platelet derived growth factor and a tryptophan hydroxylaseinhibitor. An example of a platelet derived growth factor is imatinib.

The dose and method of administration of a TPH inhibitor can be readilydetermined by those of ordinary skill in the art. For example, aninhibitor can be titrated until the severity of a serotonin-mediatedadverse effect diminishes. Alternatively, blood 5-HT levels can bedirectly measured and correlated to the amount of TPH inhibitoradministered.

5.5. Pharmaceutical Compositions

This invention encompasses pharmaceutical compositions comprising a TPHinhibitor (e.g., a potent TPH1 inhibitor) and at least one other activepharmaceutical ingredient that can affect vasodilation orvasoconstriction. Examples of such other active pharmaceuticalingredients include those described herein, as well as other known bythose skilled in the art.

Certain pharmaceutical compositions are single unit dosage formssuitable for oral, mucosal (e.g., nasal, sublingual, vaginal, buccal, orrectal), parenteral (e.g., subcutaneous, intravenous, bolus injection,intramuscular, or intraarterial), or transdermal administration to apatient. Examples of dosage forms include, but are not limited to:tablets; caplets; capsules, such as soft elastic gelatin capsules;cachets; troches; lozenges; dispersions; suppositories; ointments;cataplasms (poultices); pastes; powders; dressings; creams; plasters;solutions; patches; aerosols (e.g., nasal sprays or inhalers); gels;liquid dosage forms suitable for oral or mucosal administration to apatient, including suspensions (e.g., aqueous or non-aqueous liquidsuspensions, oil-in-water emulsions, or a water-in-oil liquidemulsions), solutions, and elixirs; liquid dosage forms suitable forparenteral administration to a patient; and sterile solids (e.g.,crystalline or amorphous solids) that can be reconstituted to provideliquid dosage forms suitable for parenteral administration to a patient.

The formulation should suit the mode of administration. For example, theoral administration of a compound susceptible to degradation in thestomach may be achieved using an enteric coating. Similarly, aformulation may contain ingredients that facilitate delivery of theactive ingredient(s) to the site of action. For example, compounds maybe administered in liposomal formulations in order to protect them fromdegradative enzymes, facilitate transport in circulatory system, andeffect their delivery across cell membranes.

Similarly, poorly soluble compounds may be incorporated into liquiddosage forms (and dosage forms suitable for reconstitution) with the aidof solubilizing agents, emulsifiers and surfactants such as, but notlimited to, cyclodextrins (e.g., α-cyclodextrin, β-cyclodextrin,Captisol®, and Encapsin™ (see, e.g., Davis and Brewster, Nat. Rev. DrugDisc. 3:1023-1034 (2004)), Labrasol®, Labrafil®, Labrafac®, cremafor,and non-aqueous solvents, such as, but not limited to, ethyl alcohol,isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol,benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, dimethyl sulfoxide (DMSO), biocompatible oils (e.g.,cottonseed, groundnut, corn, germ, olive, castor, and sesame oils),glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols, fatty acidesters of sorbitan, and mixtures thereof (e.g., DMSO: cornoil).

Poorly soluble compounds may also be incorporated into suspensions usingother techniques known in the art. For example, nanoparticles of acompound may be suspended in a liquid to provide a nanosuspension (see,e.g., Rabinow, Nature Rev. Drug Disc. 3:785-796 (2004)). Nanoparticleforms of compounds described herein may be prepared by the methodsdescribed in U.S. Patent Publication Nos. 2004-0164194, 2004-0195413,2004-0251332, 2005-0042177 A1, 2005-0031691 A1, and U.S. Pat. Nos.5,145,684, 5,510,118, 5,518,187, 5,534,270, 5,543,133, 5,662,883,5,665,331, 5,718,388, 5,718,919, 5,834,025, 5,862,999, 6,431,478,6,742,734, 6,745,962, the entireties of each of which are incorporatedherein by reference. In one embodiment, the nanoparticle form comprisesparticles having an average particle size of less than about 2000 nm,less than about 1000 nm, or less than about 500 nm.

The composition, shape, and type of a dosage form will typically varydepending with use. For example, a dosage form used in the acutetreatment of a disease may contain larger amounts of one or more of theactive ingredients it comprises than a dosage form used in the chronictreatment of the same disease. Similarly, a parenteral dosage form maycontain smaller amounts of one or more of the active ingredients itcomprises than an oral dosage form used to treat the same disease. Howto account for such differences will be apparent to those skilled in theart. See, e.g., Remington's Pharmaceutical Sciences, 18th ed., MackPublishing, Easton Pa. (1990).

5.5.1. Oral Dosage Forms

Pharmaceutical compositions of the invention suitable for oraladministration can be presented as discrete dosage forms, such as, butare not limited to, tablets (e.g., chewable tablets), caplets, capsules,and liquids (e.g., flavored syrups). Such dosage forms containpredetermined amounts of active ingredients, and may be prepared bymethods of pharmacy well known to those skilled in the art. Seegenerally, Remington's Pharmaceutical Sciences, 18th ed., MackPublishing, Easton Pa. (1990).

Typical oral dosage forms are prepared by combining the activeingredient(s) in an intimate admixture with at least one excipientaccording to conventional pharmaceutical compounding techniques.Excipients can take a wide variety of forms depending on the form ofpreparation desired for administration.

Because of their ease of administration, tablets and capsules representthe most advantageous oral dosage unit forms. If desired, tablets can becoated by standard aqueous or non-aqueous techniques. Such dosage formscan be prepared by conventional methods of pharmacy. In general,pharmaceutical compositions and dosage forms are prepared by uniformlyand intimately admixing the active ingredients with liquid carriers,finely divided solid carriers, or both, and then shaping the productinto the desired presentation if necessary. Disintegrants may beincorporated in solid dosage forms to facility rapid dissolution.Lubricants may also be incorporated to facilitate the manufacture ofdosage forms (e.g., tablets).

5.5.2. Parenteral Dosage Forms

Parenteral dosage forms can be administered to patients by variousroutes including subcutaneous, intravenous (including bolus injection),intramuscular, and intraarterial. Because their administration typicallybypasses patients' natural defenses against contaminants, parenteraldosage forms are specifically sterile or capable of being sterilizedprior to administration to a patient. Examples of parenteral dosageforms include solutions ready for injection, dry products ready to bedissolved or suspended in a pharmaceutically acceptable vehicle forinjection, suspensions ready for injection, and emulsions.

Suitable vehicles that can be used to provide parenteral dosage forms ofthe invention are well known to those skilled in the art. Examplesinclude: Water for Injection USP; aqueous vehicles such as SodiumChloride Injection, Ringer's Injection, Dextrose Injection, Dextrose andSodium Chloride Injection, and Lactated Ringer's Injection;water-miscible vehicles such as ethyl alcohol, polyethylene glycol, andpolypropylene glycol; and non-aqueous vehicles such as corn oil,cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropylmyristate, and benzyl benzoate.

6. EXAMPLES 6.1. Production of tph1 Gene Disrupted Mice

Exon 3 of the murine TPH1 gene was removed by gene targeting essentiallyas described by Wattler et al., Biotechniques 26(6):1150-6 (1999). Theresulting knockout animals displayed normal TPH activity in the brainbut drastically reduced TPH expression in the gut.

6.2. Physiological Effects of tph1 Gene Disruption

Mice homozygous (−/−) for the disruption of tph1 were studied inconjunction with mice heterozygous (+/−) for the disruption of the gene,along with wild-type (+/+) litter mates. During this analysis, the micewere subject to a medical work-up using an integrated suite of medicaldiagnostic procedures designed to assess the function of the major organsystems in a mammalian subject. By studying the homozygous (−/−)knockout mice in the described numbers and in conjunction withheterozygous (+/−) and wild-type (+/+) litter mates, more reliable andrepeatable data was obtained.

Disruption of tph1 gene primarily affected the GI tract isoform of TPH(TPH1), and had little or no effect on the brain isoform of TPH (TPH2).Disruption of the gene caused no measurable adverse effects on thecentral nervous system. This was confirmed by serotonin immunochemistry,which showed that serotonin was greatly reduced or absent in thestomach, duodenum, jejunum, ileum, cecum and colon, while serotoninlevels were unaffected in raphe neurons.

Mice homozygous (−/−) for the disruption of the tph1 gene had a decreasein thrombosis without a significant increase in bleeding or otheradverse indications.

6.3. HPLC Characterization

In some of the following synthetic examples, high performance liquidchromatography (HPLC) retention times are provided. Unless otherwisenoted, the various conditions used to obtain those retention times aredescribed below:

Method A: YMC-PACK ODS-A 3.0×50 mm; Solvent A=90% water, 10% MeOH, 0.1%TFA; Solvent B=90% MeOH, 10% water, 0.1% TFA; B % from 0 to 100% over 4min.; flow rate=2 ml/min; observation wavelength=220 nm.

Method B: YMC-PACK ODS-A 3.0×50 mm; Solvent A=90% water, 10% MeOH, 0.1%TFA; Solvent B=90% MeOH, 10% water, 0.1% TFA; % B from 10 to 100% over 4min.; flow rate=3 ml/min; observation wavelength=220 nm.

Method C: YMC-PACK ODS-A 3.0×50 mm; Solvent A=90% water, 10% MeOH, 0.1%TFA; Solvent B=90% MeOH, 10% water, 0.1% TFA; B % from 0 to 100% over 5min.; flow rate=2 ml/min.; observation wavelength=220 nm.

Method D: Shim VP ODS 4.6×50 mm; Solvent A=90% water, 10% MeOH, 0.1%TFA; Solvent B=90% MeOH, 10% water, 0.1% TFA; B % from 0 to 100% over 4min.; flow rate=3 ml/min.; observation wavelength=220 nm.

Method E: Shim VP ODS 4.6×50 mm; Solvent A=90% water, 10% MeOH, 0.1%TFA; Solvent B=90% MeOH, 10% water, 0.1% TFA; B % from 0 to 100% over 4min.; flow rate=3 ml/min; observation wavelength=254 nm.

Method F: YMC-PACK ODS-A 4.6×33 mm; Solvent A=90% water, 10% MeOH, 0.1%TFA; Solvent B=90% MeOH, 10% water, 0.1% TFA; B % from 0 to 100% over 4min.; flow rate=3 ml/min.; observation wavelength=220 nm.

Method G: YMC-PACK ODS-A 4.6×50 mm; Solvent A=90% water, 10% MeOH, 0.1%TFA; Solvent B=90% MeOH, 10% water, 0.1% TFA; B % from 0 to 100% over 2min.; flow rate=2.5 ml/min.; observation wavelength=220 nm.

Method H: C18 4.6×20 mm; Solvent A=90% water, 10% MeOH, 0.1% TFA;Solvent B=90% MeOH, 10% water, 0.1% TFA; B % from 0 to 100% over 2 min.flow rate=2 ml/min.; observation wavelength=220 nm.

Method I: YMC PACK ODS-A 3.0×50 mm; Solvent A=90% water, 10% MeOH, 0.1%TFA; Solvent B=90% MeOH, 10% water, 0.1% TFA; B % from 10 to 100% over 4min.; flow rate=2 ml/min.; observation wavelength=220 nm.

Method J: YMC Pack ODS-A 3.0×50 mm; Solvent A=H₂O, 0.1% TFA; SolventB=MeOH, 0.1% TFA; % B from about 10 to about 90% over 4 min.; flowrate=2 ml/min.; observation wavelength=220 nm.

Method K: Sunfire C18 50 mm×4.6 mm×3.5 μm; Solvent A=10 mM NH₄OAc inwater; Solvent B=MeCN; B % from 10 to 95% over 2 min.; flow rate=4.5ml/min.; observation wavelength=220 nm.

Method L: Sunfire C18 50 mm×4.6 mm×3.5 μm; Solvent A=10 mM NH₄OAc;Solvent B=MeCN; B % from 2 to 20% over 0.8 min, then to 95% B over 2min; flow rate=4.5 ml/min.; observation wavelength=220 nm.

Method M: YMC-PACK ODS-A 4.6×33 mm; Solvent A=90% water, 10% MeOH, 0.1%TFA; Solvent B=90% MeOH, 10% water, 0.1% TFA; B % from 0 to 100% over 5min.; flow rate=2.5 ml/min.; observation wavelength=254 nm.

Method N: YMC-PACK ODS-A 3.0×50 mm; Solvent A=H₂O, 0.1% TFA; SolventB=MeOH, 0.1% TFA; B % from 10 to 90% over 4 min.; flow rate=2 ml/min.;observation wavelength=220 and 254 nm.

Method O: YMC-PACK ODS-A 3.0×50 mm; Solvent A=90% water, 10% MeOH with0.1% TFA; Solvent B=90% MeOH, 10% water with 0.1% TFA; B % from 0 to100% over 4 min.; flow, rate=2 ml/min.; observation wavelength=220 and254 nm.

Method P: ShimPack VP ODS 4.6×50 mm; Solvent A=90% H₂O, 10% MeOH, 1%TFA; Solvent B=10% H₂O, 90% MeOH, 1% TFA; B % from 0 to 100% over 2min.; flow rate=3.5 ml/min.; observation wavelength=220 and 254 nm.

Method Q: Shim VP ODS 4.6×50 mm; Solvent A=H₂O with 0.1% TFA; SolventB=MeOH with 0.1% TFA; B % from 0 to 100% over 4 min.; flow rate=3ml/min.; observation wavelength=254 nm.

Method R: YMC Pack ODS-A 4.6×33 mm; Solvent A=H₂O, 0.1% TFA; SolventB=MeOH with 0.1% TFA; B % from 10 to 90% over 3 min.; flow rate 2ml/min.; observation wavelength 220 and 254 nm.

Method S: YMC-Pack ODS-A 3.0×50 mm; Solvent A=90% H₂O, 10% MeOH, 1% TFA;Solvent B=10% H₂O, 90% MeOH, 1% TFA; B % from 10 to 90% over 4 min.;flow rate=2 ml/min. observation wavelength=220 and 254 nm.

6.4. Synthesis of(S)-2-Amino-3-(4-(4-amino-6-((R)-1-(naphthalen-2-yl)ethylamino)-1,3,5-triazin-2-yl)phenyl)propanoicacid

A mixture of 2-amino-4,6-dichloro-[1,3,5]triazine (200 mg, 1.21 mmol),(R)-(+)-1-(2-naphthyl)ethylamine (207 mg, 1.21 mmol) anddiisopropyl-ethylamine (3.63 mmol) was dissolved in 150 ml of1,4-dioxane. The solution was refluxed at 90° C. for 3 hours. After thecompletion of reaction (monitored by LCMS), solvent was removed and thereaction mixture was extracted with CH₂Cl₂ (100 ml) and H₂O (100 ml).The organic layer was separated and washed with H₂O (2×100 ml), driedover Na₂SO₄, and concentrated in vacuo to give crude intermediate. Thecrude compound was dissolved in 5 ml of MeCN and 5 ml of H₂O in a 20 mlmicrowave reaction vial. To this solution were addedL-p-borono-phenylalanine (253 mg, 1.21 mmol), sodium carbonate (256 mg,2.42 mmol) and catalytic amount ofdichlorobis(triphenylphosphine)-palladium(II) (42.1 mg, 0.06 mmol). Themixture was sealed and stirred in the microwave reactor at 150° C. for 5minutes, followed by the filtration through celite. The filtrate wasconcentrated and dissolved in MeOH and H₂O (1:1) and purified bypreparative HPLC using MeOH/H₂O/TFA solvent system. The combined purefractions were evaporated in vacuo and further dried on a lyophilizer togive 238 mg of2-amino-3-{4-[4-amino-6-(1-naphthalen-2-yl)-ethylamino)-[1,3,5]triazin-2-yl]-phenyl}-propionicacid (yield: 46%, LC: Column: YMC Pack ODS-A 3.0×50 mm, % B=0˜100%,Gradient time=4 min, Flow Rate=2 ml/min, wavelength=220, Solvent A=90:10water:MeOH w/0.1% TFA, Solvent B=90:10 MeOH:water w/0.1% TFA, RT=2.785min, MS: M+1=429). NMR: ¹H-NMR (400 MHz, CD₃OD): δ 1.65 (d, 3H),3.22-3.42 (m, 2H), 4.3 (m, 1H), 5.45 (m, 1H), 7.4 (m, 1H), 7.6 (m 4H),7.8 (m, 4H), 8.2 (m, 2H).

6.5. Alternative Synthesis of(S)-2-Amino-3-(4-(4-amino-6-((R)-1-(naphthalen-2-yl)ethylamino)-1,3,5-triazin-2-yl)phenyl)propanoicacid

(R)-1-(1-(Napthalen-2-yl)ethyl) cyanoguanidine was prepared by forming amixture of naphthalene amine (1 equivalent), sodium dicyanide (0.95 eq.)and followed by 5N HCl (1 eq.) in n-BuOH: H₂O (1:1). The mixture wasrefluxed for 1 day in a sealed tube at 160° C., and progress of reactionwas monitored by LCMS. After completion of reaction, solvent (n-BuOH)was removed under reduced pressure and 1N HCl was added to adjust pH to3-5 range. The aqueous solution was extracted with EtOAc (2×100) andcombined organic phase was dried over Na₂SO₄. Solvent was removed invacuo to give crude product. The compound was purified by ISCO columnchromatography using as the solvent system EtOAc:hexane (7:3 and 1:1),to obtain white solid 48-71% yield for 1 g to 22.5 gram scale. NMR:¹H-NMR (400 MHz, CD₃OD): δ 1.5 (d, 3H), 5.1 (m, 1H), 7.5 (m, 4H), 7.8(s, 1H), 7.9 (m, 2H); LCMS: RT 1.69, M+1: 239, Yield: 71%.

The title compound was prepared from (R)-1-(1-(napthalen-2-yl)ethyl)cyanoguanidine according to the method shown in Scheme 6.

6.6. Synthesis of(S)-2-Amino-3-(4-(4-amino-6-((4′-methylbiphenyl-4-yl)methylamino)-1,3,5-triazin-2-yl)phenyl)propanoicacid

A mixture of 2-amino-4,6-dichloro-[1,3,5]triazine (100 mg, 0.606 mmol),4′-methyl-biphenyl-4-yl-methylamine (142 mg, 0.606 mmol), and cesiumcarbonate (394 mg, 1.21 mmol) was dissolved in 1,4-dioxane (1.5 ml) andH₂O (1.5 ml) in a 5 ml microwave vial. The mixture was stirred inmicrowave reactor at 100° C. for 15 minutes. Solvent was removed and theresidue was dissolved in CH₂Cl₂ (20 ml) and washed with H₂O (2×20 ml),dried over Na₂SO₄ and then removed in vacuo. The crude intermediate wasthen dissolved in 1.5 ml of MeCN and 1.5 ml of H₂O in a 5 ml microwavevial. To this solution were added L-p-borono-phenylalanine (126 mg,0.606 mmol), sodium carbonate (128 mg, 1.21 mmol) and catalytic amountof dichlorobis(triphenylphosphine)-palladium(II) (21.1 mg, 0.03 mmol).The mixture was sealed and stirred in the microwave reactor at 150° C.for 5 minutes followed by the filtration through celite. The filtratewas concentrated and dissolved in MeOH and H₂O (1:1) and purified bypreparative HPLC using MeOH/H₂O/TFA solvent system. The combined purefractions were evaporated in vacuo and further dried on a lyophilizer togive 21.6 mg of2-amino-3-(4-{4-amino-6-[(4′-methyl-biphenyl-4-ylmethyl)-amino]-[1,3,5]triazin-2-yl}-phenyl)-propionicacid (LC: Column: YMC Pack ODS-A 3.0×50 mm, % B=0˜100%, Gradient time=4min, Flow Rate=2 ml/min, wavelength=220, Solvent A=90:10 water:MeOH w/0.1% TFA, Solvent B=90:10 MeOH:water w/0.1% TFA, RT=3.096 min, MS:M+1=455). ¹H NMR (400 MHz, CD₃OD) δ 2.33 (s, 3H), 3.24-3.44 (m, 2H),4.38 (m, 1H), 7.02 (d, 2H), 7.42 (m, 2H), 7.50-7.60 (m, 6H), 8.22 (m,2H).

6.7. Synthesis of(S)-2-Amino-3-(4-(4-morpholino-6-(naphthalen-2-ylmethylamino)-1,3,5-triazin-2-yl)phenyl)propanoicacid

A mixture of 2,4-dichloro-6-morpholin-4-yl-[1,3,5]triazine (121 mg,0.516 mmol), C-naphthalen-2-yl-methylamine hydrochloride (100 mg, 0.516mmol), cesium carbonate (336 mg, 1.03 mmol) was dissolved in 1,4-Dioxane(1.5 ml) and H₂O (1.5 ml) in a 5 ml microwave vial. The mixture wasstirred in microwave reactor at 180° C. for 600 seconds. Solvent wasremoved, and the residue was dissolved in CH₂Cl₂ (10 ml) and washed withH₂O (2×10 ml), dried over Na₂SO₄ and then in vacuo. The residue waspurified by preparative HPLC to give 20 mg intermediate (yield 11%,M+1=356). The intermediate was then dissolved in 0.5 ml of MeCN and 0.5ml of H₂O in a 2 ml microwave vial. To this solution were addedL-p-borono-phenylalanine (11.7 mg, 0.0562 mmol), sodium carbonate (11.9mg, 0.112 mmol) and a catalytic amount ofdichlorobis(triphenylphosphine)-palladium(II) (2.0 mg, 5%). The mixturewas sealed and stirred in the microwave reactor at 150° C. for 5 minutesfollowed by the filtration through celite. The filtrate was concentratedand dissolved in MeOH and H₂O (1:1) and purified by preparative HPLCusing MeOH/H₂O/TFA solvent system. The combined pure fractions wereevaporated in vacuo and further dried on lyophilizer to give 17 mg of2-amino-3-(4-{4-morpholin-4-yl-6-[(naphthalene-2-ylmethyl)-amino]-[1,3,5]triazin-2-yl}-phenyl)-propionicacid (yield: 63%, LC: Method B, RT=3.108 min, MS: M+1=486).

6.8. Synthesis of(2S)-2-Amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(2-(trifluoromethyl)phenyl)ethoxy)pyrimidin-4-yl)phenyl)propanoicacid

Tetrabutylammonium fluoride (0.1 ml; 1.0 M solution in tetrahydrofuran)was added to a solution of 2-trifluoromethyl-benzaldehyde (1.74 g, 10mmol) and trifluoromethyltrimethylsilane (TMSCF₃) (1.8 ml, 12 mmol) in10 ml THF at 0° C. The formed mixture was warmed up to room temperatureand stirred for 4 hours. The reaction mixture was then treated with 12ml of 1N HCl and stirred overnight. The product was extracted with ethylacetate (3×20 ml). The organic layer was separated and dried over sodiumsulfate. The organic solvent was evaporated to give 2.2 g of1-(2-trifluoromethylphenyl)-2,2,2-trifluoro-ethanol, yield 90%.

NaH (80 mg, 60%, 3.0 mmol) was added to a solution of1-(2-trifluoromethylphenyl)-2,2,2-trifluoro-ethanol (244 mg, 1 mmol) in10 ml of anhydrous THF. The mixture was stirred for 20 minutes,2-amino-4,6-dichloro-pyrimidine (164 mg, 1 mmol) was added and then thereaction mixture was heated at 70° C. for 1 hour. After cooling, 5 mlwater was added and ethyl acetate (20 ml) was used to extract theproduct. The organic layer was dried over sodium sulfate. The solventwas removed by rotovap to give 267 mg of4-chloro-6-[2,2,2-trifluoro-1-(2-trifluoromethylphenyl)-ethoxy]-pyrimidin-2-ylamine,yield 71%.

In a microwave vial,4-chloro-2-amino-6-[1-(2-trifluoromethylphenyl)-2,2,2-trifluoro-ethoxy]-pyrimidine(33 mg, 0.1 mmol), 4-borono-L-phenylalanine (31 mg, 0.15 mmol) and 1 mlof acetonitrile, 0.7 ml of water. 0.3 ml of 1N aqueous sodium carbonatewas added to above solution followed by 5 mole percent ofdichlorobis(triphenylphosphine)-palladium(II). The reaction vessel wassealed and heated at 150° C. for 5 minutes with microwave irradiation.After cooling, the reaction mixture was evaporated to dryness. Theresidue was dissolved in 2.5 ml of methanol, and then was purified byPrep-LC to give 5.6 mg of2-amino-3-(4-{2-amino-6-[2,2,2-trifluoro-1-(2-triifluoromethylphenyl)-ethoxy]-pyrimidin-4-yl}-phenyl)-propionicacid. ¹H NMR (400 MHz, CD₃OD) δ 7.96 (m, 3H), 7.80 (d, J=8.06 Hz, 1H),7.74 (t, J=7.91 Hz 1H), 7.63 (t, J=8.06 Hz, 1H), 7.41 (d, J=8.3 Hz, 2H),7.21 (m, 1H), 6.69 (s, 1H), 3.87 (m, 1H), 3.34 (m, 1H), 3.08 (m, 1H).

6.9. Synthesis of(2S)-2-Amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-p-tolylethoxy)pyrimidin-4-yl)phenyl)propanoicacid

Tetrabutylammonium fluoride (0.1 ml; 1.0 M solution in tetrahydrofuran)was added to a solution of 4-methyl-benzaldehyde (1.2 g, 10 mmol) andTMSCF₃ (1.8 ml, 12 mmol) in 10 ml THF at 0° C. The formed mixture waswarmed up to room temperature and stirred for 4 hours. The reactionmixture was then treated with 12 ml of 1N HCl and stirred overnight. Theproduct was extracted with ethyl acetate (3×20 ml). The organic layerwas separated and dried over sodium sulfate. The organic solvent wasevaporated to give 1.6 g of 1-(4-methylphenyl)-2,2,2-trifluoro-ethanol,yield 86%.

NaH (80 mg, 60%, 3.0 mmol) was added to a solution of1-(4-methylphenyl)-2,2,2-trifluoro-ethanol (190 mg, 1 mmol) in 10 ml ofanhydrous THF. The mixture was stirred for 20 minutes,2-amino-4,6-dichloro-pyrimidine (164 mg, 1 mmol) was added and then thereaction mixture was heated at 70° C. for 1 hour. After cooling, 5 mlwater was added and ethyl acetate (20 ml) was used to extract theproduct. The organic layer was dried over sodium sulfate. The solventwas removed by rotovap to give 209 mg of4-chloro-6-[1-(4-methylphenyl)-2,2,2-trifluoro-ethoxy]-pyrimidin-2-ylamine,yield 66%.

A microwave vial was charged with4-chloro-2-amino-6-[1-(4-methylphenyl)-2,2,2-trifluoro-ethoxy]-pyrimidine(33 mg, 0.1 mmol), 4-borono-L-phenylalanine (31 mg, 0.15 mmol) and 1 mlof acetonitrile, 0.7 ml of water. Aqueous sodium carbonate (0.3 ml, 1N)was added to above solution followed by 5 mol percent ofdichlorobis(triphenylphosphine)-palladium(II). The reaction vessel wassealed and heated to 150° C. for 5 minutes with microwave. Aftercooling, the reaction mixture was evaporated to dryness. The residue wasdissolved in 2.5 ml of methanol, was then purified by Prep-LC to give14.6 mg of2-amino-3-(4-{2-amino-6-[2,2,2-trifluoro-1-(4-methylphenyl)-ethoxy]-pyrimidin-4-yl}-phenyl)-propionicacid. ¹H NMR (300 MHz, CD₃OD) δ 7.94 (d, J=8.20 Hz, 2H), 7.47 (d, J=7.24Hz, 4H), 7.27 (d, J=8.01 Hz, 2H) 6.80 (s, 1H), 6.75 (m, 1H), 4.30 (t,1H), 3.21-3.44 (m, 2H), 2.37 (s, 3H).

6.10. Synthesis of(2S)-2-Amino-3-(4-(2-amino-6-(1-cyclohexyl-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoicacid

Cyclohexanecarbaldehyde (0.9 g, 5 mmol) was dissolved in 10 ml aqueous1,4-dioxane, to which 200 mg (10 mmol) sodium borohydride was added. Thereaction was run overnight at room temperature. After completion of thereaction, 5 ml 10% HCl solution was added and the product was extractedwith ethyl acetate. The organic layer was separated and dried oversodium sulfate. The organic solvent was evaporated to give 0.8 g of1-cyclohexyl-2,2,2-trifluoro-ethanol, yield 88%.

NaH (80 mg, 60%, 3.0 mmol) was added to the solution of1-cyclohexyl-2,2,2-trifluoro-ethanol (182 mg, 1 mmol) in 10 ml ofanhydrous THF, the mixture was stirred for 20 minutes,2-amino-4,6-dichloro-pyrimidine (164 mg, 1 mmol) was added and then thereaction mixture was heated at 70° C. for 1 hour. After cooling, 5 mlwater was added and ethyl acetate (20 ml) was used to extract theproduct. The organic layer was dried over sodium sulfate. The solventwas removed by rotovap to give 202 mg of4-chloro-6-[1-cyclohexyl-2,2,2-trifluoro-ethoxy]-pyrimidin-2-ylamine,yield 65%.

In a microwave vial,4-chloro-2-amino-6-[1-cyclohexane-2,2,2-trifluoro-ethoxy]-pyrimidine (33mg, 0.1 mmol), 4-borono-L-phenylalanine (31 mg, 0.15 mmol) and 1 ml ofacetonitrile, 0.7 ml of water, 0.3 ml of aqueous sodium carbonate (1M)was added to above solution followed by 5 mol percent ofdichlorobis(triphenylphosphine)-palladium(II). The reaction vessel wassealed and heated to 150° C. for 5 minutes with a microwave. Aftercooling, the reaction mixture was evaporated to dryness, the residue wasdissolved in 2.5 ml of methanol, and the product was purified by Prep-LCto give 4.9 mg2-amino-3-{4-[2-amino-6-(1-cyclohexyl-2,2,2-trifluoro-ethoxy]-pyrimidin-4-yl}-phenyl)-propionicacid. ¹H NMR (300 MHz, CD₃Cl) δ 7.95 (d, J=8.39 Hz, 2H), 7.49 (d, J=8.39Hz, 2H), 6.72 (s, 1H), 5.90 (m, 1H), 4.33 (t, 1H), 3.21-3.44 (m, 2H),1.73-2.00 (m, 6H), 1.23-1.39 (m, 5H).

6.11. Synthesis of(S)-2-Amino-3-(4-(6-(2-fluorophenoxy)pyrimidin-4-yl)phenyl)propanoicacid

NaH (80 mg, 60%, 3.0 mmol) was added to a solution of 2-fluorophenol(112 mg, 1 mmol) in 10 ml of anhydrous THF, the mixture was stirred for20 minutes, 4,6-dichloro-pyrimidine (149 mg, 1 mmol) was added and thenthe reaction mixture was heated at 70° C. for 1 hour. After cooling, 5ml water was added and ethyl acetate (20 ml) was used to extract theproduct. The organic layer was dried over sodium sulfate. The solventwas removed by rotovap to give 146 mg of4-chloro-6-(2-fluorophenoxy)-pyrimidine, yield 65%.

A microwave vial (2 ml) was charged with4-chloro-6-[2-fluorophenoxy]-pyrimidine, (33 mg, 0.1 mmol),4-borono-L-phenylalanine (31 mg, 0.15 mmol) and 1 ml of actonitrile, 0.7ml of water, 0.3 ml of aqueous sodium carbonate (1M) was added to abovesolution followed by 5 mol % ofdichlorobis(triphenylphosphine)-palladium(II). The reaction vessel wassealed and heated to 150° C. for 5 minutes by microwave. After cooling,the reaction mixture was evaporated to dryness, the residue wasdissolved in 2.5 ml of methanol, and the product was purified withPrep-LC to give 4.9 mg2-amino-3-{4-[2-amino-6-(1-2-fluorophenyl-2,2,2-trifluoro-ethoxy]-pyrimidin-4-yl}-phenyl)-propionicacid. ¹H NMR (400 MHz, CD₃OD) δ 8.74 (s, 1H), 8.17 (d, J=8.06 Hz, 2H),7.63 (s, 1H), 7.50 (d, J=8.06 Hz, 2H), 7.30 (m, 5H), 4.33 (m, 1H), 3.34(m, 1H).

6.12. Synthesis of(2S)-2-Amino-3-(4-(4-(3-(4-chlorophenyl)piperidin-1-yl)-1,3,5-triazin-2-yl)phenyl)propanoicacid

3-(4-Chlorophenyl)piperidine (232 mg, 1 mmol) was added to a solution of2,4-dichlorotriazine (149.97 mg, 1 mmol), and 300 mg diisopropylethylamine in 10 ml THF at 0° C. The formed mixture was warmed up to roomtemperature and stirred for 1 hour. The product was extracted with ethylacetate (3×20 ml). The organic layer was separated and dried over sodiumsulfate. The organic solvent was evaporated to give 328 mg of2-chloro-4-[3-(4-chlorophenyl)-piperidin-1-yl]-[1,3,5]triazine.

A microwave vial was charged with2-chloro-4-[3-(4-chlorophenyl)-piperidin-1-yl]-[1,3,5]triazine (62 mg,0.2 mmol), 4-borono-L-phenylalanine (60 mg, 0.3 mmol), 1 ml ofacetonitrile, and 0.7 ml of water. Aqueous sodium carbonate (0.6 ml; 1M)was added to the solution, followed by 5 mol percentdichlorobis(triphenylphosphine)-palladium(II). The reaction vessel wassealed and heated to 150° C. for 5 minutes with microwave. Aftercooling, the reaction mixture was evaporated to dryness. The residue wasdissolved in 2.5 ml of methanol, was then purified by Prep-LC to give5.1 mg of2-amino-3-(4-{4-[3-(4-chlorophenyl)-piperidin-1-yl]-[1,3,5]triazin-2-yl}-phenyl)-propionicacid. ¹H NMR (400 MHz, CD₃Cl) δ 8.58 (d, 2H), 8.05 (d, 2H), 7.47 (m,5H), 4.96 (m, 1H), 4.23 (m, 2H), 3.21-3.44 (m, 4H), 2.37 (m, 5H).

6.13. Synthesis of(2S)-2-Amino-3-(4-(4-amino-6-(2,2,2-trifluoro-1-phenylethoxy)-1,3,5-triazin-2-yl)phenyl)propanoicacid

NaH (80 mg, 60%, 3.0 mmol) was added to a solution of2,2,2-trifluoro-1-phenylethanol (176 mg, 1 mmol) in 10 ml of anhydrous1,4-dioxane. The mixture was stirred for 20 minutes, then added to asolution of 2-amino-4,6-dichloro-triazine (164 mg, 1 mmol) in 30 ml of1,4-dioxane at 0° C. for 1 hour. The reaction mixture was then warmed toroom temperature. After completion of the reaction, 5 ml of water wasadded and ethyl acetate (20 ml) was used to extract the product. Theorganic layer was dried over sodium sulfate. The solvent was removed byrotovap to give 198 mg of4-chloro-6-[2,2,2-trifluoro-1-phenyl-ethoxy]-[1,3,5]triazine-2-ylamine,yield 65%.

A microwave vial was charged with4-chloro-6-[2,2,2-trifluoro-1-phenyl-ethoxy]-[1,3,5]triazine-2-ylamine(33 mg, 0.1 mmol), 4-borono-L-phenylalanine (31 mg, 0.15 mmol), 1 ml ofactonitrile, and 0.7 ml of water. Aqueous sodium carbonate (0.3 ml, 1M)was added to above solution followed by 5 mol percentdichlorobis(triphenylphosphine)-palladium(II). The reaction vessel wassealed and heated to 150° C. for 5 minutes by microwave. After cooling,the reaction mixture was evaporated to dryness. The residue wasdissolved in 2.5 ml of methanol, was then purified with Prep-LC to give3.2 mg2-amino-3-{4-[4-amino-6-(1-phenyl-2,2,2-trifluoro-ethoxy]-[1,3,5]triazin-2-yl]-phenyl)-propionicacid. ¹H NMR (300 MHz, CD₃OD) δ 8.22 (d, J=8.20 Hz, 2H), 7.52 (m, 2H),7.33 (m, 5H) 6.62 (m, 1H), 4.19 (t, 1H), 3.1-3.33 (m, 2H).

6.14. Synthesis of(S)-2-Amino-3-(5-(4-amino-6-((R)-1-(naphthalen-2-yl)ethylamino)-1,3,5-triazin-2-yl)pyridin-2-yl)propanoicacid

A microwave vial was charged with6-chloro-N-[1-naphthalen-2yl-ethyl]-[1,3,5]triazine-2,4-diamine (30 mg,0.1 mmol), 2-bocprotected-amino-3-{5-[4,4,5,5,-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyridin2-yl-]-propionicacid (50 mg, 0.15 mmol) 1 ml of acetonitrile, and 0.7 ml of water.Aqueous sodium carbonate (0.3 ml; 1N) was added to the solution,followed by 5 mol percent dichlorobis(triphenylphosphine)-palladium(II).The reaction vessel was sealed and heated to 150° C. for 5 minutes bymicrowave. After cooling, the reaction mixture was evaporated todryness. The residue was dissolved in 2.5 ml of methanol, and was thenpurified by Prep-LC to give 7 mg of boc protected2-amino-3-{5-[4-amino-6-(1-naphthalen-2-yl-ethylamino)-[1,3,5]triazin-2-yl]-pyridin-2-yl}proionicacid.

The above product (7.0 mg) was dissolved in 0.1 ml of 10% TFA/DCMsolution for 2 hours to provide 1.1 mg of2-amino-3-{3-[4-amino-6-(1-naphthalen-2-yl-ethylamino)-[1,3,5]triazin-2-yl]-pyridin-2-yl}proionicacid. ¹H NMR (300 MHz, CD₃Cl) δ 9.35 (d, 1H), 8.57 (m, 1H), 7.85 (m,4H), 7.45 (m, 4H), 6.94 (s, 1H), 5.58 (m, 1H), 4.72 (m, 2H), 4.44 (m,1H), 1.42 (d, 3H).

6.15. Synthesis of(S)-2-Amino-3-(3-(4-amino-6-((R)-1-(naphthalen-2-yl)ethylamino)-1,3,5-triazin-2-yl)-1H-pyrazol-1-yl)propanoicacid

6-Chloro-N-[1-naphthalen-2-yl-ethyl]-[1,3,5]triazine-2,4-diamine (30 mg,0.1 mmol), 2-boc-protectedamino-3-{3-[4,4,5,5,-tetramethyl-[1,3,2]dioxaborolan-2-yl)-pyrazol-1-yl]-propionicacid (50 mg, 0.15 mmol), 1 ml of acetonitrile, and 0.7 ml of water.Aqueous sodium carbonate (0.3 ml and 1N) was added to a microwave vial,followed by 5 mol percent ofdichlorobis(triphenylphosphine)-palladium(II). The reaction vessel wassealed and heated to 150° C. for 5 minutes with microwave. Aftercooling, the reaction mixture was evaporated to dryness, the residue wasdissolved in 2.5 ml of methanol, and then was purified with Prep-LC togive 6.8 mg of boc protected2-amino-3-{3-[4-amino-6-(1-naphthalen-2-yl-ethylamino)[1,3,5]triazin-2-yl]-pyrazol-1-yl}proionicacid.

The above product (6.8 mg) was stirred in 0.1 ml 10% TFA/DCM solutionfor 2 hours to provide 3 mg of2-amino-3-{3-[4-amino-6-(1-naphthalen-2-yl-ethylamino)-[1,3,5]triazin-2-yl]-pyrazol-1-yl}proionicacid. ¹H NMR (300 MHz, CD₃Cl) δ 8.52 (s, 1H), 8.21 (s, 1H), 7.74 (m,4H), 7.36 (m, 3H), 5.35 (m, 1H), 4.72 (m, 2H), 4.44 (m, 1H), 1.55 (d,3H).

6.16. Synthesis of(S)-2-Amino-3-(4′-(3-(cyclopentyloxy)-4-methoxybenzylamino)biphenyl-4-yl)propanoicacid

Sodium triacetoxyl-borohydride (470 mg, 2.21 mmol) was added to asolution of 4-bromo-phenylamine (252 mg, 1.47 mmol) and3-cyclopentyloxy-4-methoxy-benzaldehyde (324 mg, 1.47 mmol) in 10 ml of1,2-dichloroethtane (DCE), 0.5 ml of HOAc was added. The mixture wasstirred overnight at room temperature, followed by addition of 15 ml ofDCE. The organic phase was washed with water and dried over sodiumsulfate. The solvent was removed by rotovap to give 656 mg of crude(4-bromo-phenyl)-(3-cyclopentyloxy-4-methoxy-benzyl)-amine. It was usedfor next step without further purification.

An Emrys process vial (2-5 ml) for microwave was charged with(4-bromo-phenyl)-(3-cyclopentyloxy-4-methoxy-benzyl)-amine (84 mg, 0.22mmol), 4-borono-L-phenylalanine (46 mg, 0.22 mmol) and 2 ml ofacetonitrile. Aqueous sodium carbonate (2 ml, 1M) was added to abovesolution, followed by 5 mol percent ofdichlorobis-(triphenylphosphine)-palladium(II). The reaction vessel wassealed and heated to 150° C. for 5 minutes by microwave. After cooling,the reaction mixture was evaporated to dryness. The residue wasdissolved in 2.5 ml of methanol and purified with Prep-LC to give 5 mgof2-amino-3-[4′-(3-cyclophentyloxy-4-methoxy-benzylamino)-biphenyl-4-yl]-propionicacid, yield 5%. ¹H-NMR (400 MHz, DMSO-d₆): δ 1.46 (m, 2H), 1.62 (m, 4H),3.01 (m, 2H), 3.64 (s, 3H), 4.14 (s, 3H), 4.66 (m, 1H), 6.61 (d, 2H),6.81 (s, 2H), 6.88 (s, 1H), 7.18 (d, 2H), 7.31 (d, 2H), 7.44 (d, 2H),7.60 (m, 1H), 8.19 (s, 3H).

6.17. Synthesis of(S)-2-Amino-3-(4-(6-(3-(cyclopentyloxy)-4-methoxybenzylamino)pyrimidin-4-yl)phenyl)propanoicacid

Sodium tiracetoxyl-borohydride (985 mg, 4.65 mmol) was added to asolution of 6-chloro-pyrimidin-4-ylamine (200 mg, 1.55 mmol) and3-cyclopentyloxy-4-methoxybenzaldehyde (682 mg, 3.1 mmol) in 25 ml ofDCE. 1 ml of HOAc was added, and the mixture was stirred overnight at50° C., followed by addition of 25 ml of DCE. The organic phase waswashed with water, and the product was purified with column (silica gel,hexane:EtOAc 5:1) to give 64 mg of(6-chloro-pyrimidin-4-yl)-(3-cyclopentyloxy-4-methoxy-benzyl)-amine,yield 12%.

An Emrys process vial (2-5 ml) for microwave was charged with(6-chloro-pyrimidin-4-yl)-(3-cyclopentyloxy-4-methoxy-benzyl)-amine (64mg, 0.19 mmol), 4-borono-L-phenylalanine (40 mg, 0.19 mmol) and 2 ml ofacetonitrile. Aqueous sodium carbonate (2 ml, 1M) was added to abovesolution followed by 5 mol percent ofdichlorobis-(triphenylphosphine)-palladium(II). The reaction vessel wassealed and heated to 150° C. for 5 minutes with microwave. Aftercooling, the reaction mixture was evaporated to dryness. The residue wasdissolved in 2.5 ml of methanol and purified with Prep-LC to give 5.3 mgof2-amino-3-{4-[6-(3-cyclopentyloxy-4-methoxy-benzylamino)-pyrimidin-4-yl]-phenyl}-propionicacid, yield 6%. ¹H-NMR (400 MHz, DMSO-d₆): δ 1.46 (m, 2H), 1.62 (m, 4H),3.01 (m, 2H), 3.08 (m, 2H), 3.65 (s, 3H), 4.20 (m, 1H), 4.46 (d, 2H),4.68 (m, 1H), 6.82 (t, 2H), 6.87 (d, 2H), 7.40 (d, 2H), 7.90 (s, 2H),8.25 (s, 2H), 8.6 (s, 1H).

6.18. Synthesis of(S)-2-Amino-3-(4-(6-(3-(cyclopentyloxy)-4-methoxybenzylamino)pyrazin-2-yl)phenyl)propanoicacid

Sodium triacetoxyl-borohydride (1315 mg, 6.2 mmol) was added to asolution of 6-chloro-pyrazin-2-yl-amine (400 mg, 3.10 mmol) and3-cyclopentyloxy-4-methoxybenzaldehyde (818 mg, 3.7 mmol) in 50 ml ofDCE, 1 ml of HOAc was added and the mixture was stirred overnight at 50°C., followed by addition of another 50 ml of DCE. The organic phase waswashed with water, and the product was purified with column (silica gel,hexane:EtOAc 6:1) to give 50 mg of(6-chloro-pyrazin-2-yl)-(3-cyclopentyloxy-4-methoxy-benzyl)-amine, yield10%.

An Emrys process vial (2-5 ml) for microwave was charged with(6-chloro-pyrazin-2-yl)-(3-cyclopentyloxy-4-methoxy-benzyl)-amine (50mg, 0.15 mmol), 4-borono-L-phenylalanine (31 mg, 0.15 mmol) and 2 ml ofacetonitrile. Aqueous sodium carbonate (2 ml, 1M) was added to thesolution followed by 5 mol percent ofdichlorobis(triphenylphosphine)-palladium(II). The reaction vessel wassealed and heated to 150° C. for 5 minutes by microwave. After cooling,the reaction mixture was evaporated to dryness. The residue wasdissolved in 2.5 ml of methanol, and the product was purified withPrep-LC to give 5.5 mg of2-amino-3-{4-[6-(3-cyclopentyloxy-4-methoxy-benzylamino)-pyrazin-2-yl]-phenyl}-propionicacid, yield 6%. ¹H-NMR (400 MHz, DMSO-d₆): δ 1.46 (m, 2H), 1.62 (m, 4H),3.01 (m, 2H), 3.08 (m, 2H), 3.65 (s, 3H), 4.0 (m, 1H), 4.45 (d, 2H),4.65 (m, 1H), 6.90 (s, 2H), 6.95 (s, 1H), 7.32 (d, 2H), 7.60 (t, 1H),7.90 (s, 1H), 7.95 (d, 2H), 8.25 (s, 1H).

6.19. Synthesis of(S)-2-Amino-3-(4-(5-((4′-methylbiphenyl-2-yl)methylamino)pyrazin-2-yl)phenyl)propanoicacid

Sodium tiracetoxyl borohydride (215 mg, 1.02 mmol) was added to thesolution of 4′-methyl-biphenyl-2-carbaldehyde and5-bromo-pyrazin-2-ylamine in 5 ml of DCE, 0.1 ml of HOAc was added andthe mixture was stirred overnight at room temperature, followed byaddition of 5 ml of DCE. The organic phase was washed with water, andpurified with column (silica gel, hexane:EtOAc 6:1) to give 100 mg of(5-bromo-pyrazin-2-yl)-(4′-methyl-biphenyl-2-ylmethyl)-amine, yield 55%.

An Emrys process vial (2-5 ml) for microwave was charged with(5-bromo-pyrazin-2-yl)-(4′-methyl-biphenyl-2-ylmethyl)-amine (25 mg,0.071 mmol), 4-borono-L-phenylalanine (22 mg, 0.11 mmol) and 1 ml ofacetonitrile. Aqueous sodium carbonate (1 ml, 1M) was added to thesolution followed by 5 mol percentdichlorobis(triphenylphosphine)-palladium(II). The reaction vessel wassealed and heated to 150° C. for 5 minutes by microwave. After cooling,the reaction mixture was evaporated to dryness. The residue wasdissolved in 2.5 ml of methanol, and the product was purified withPrep-LC to give 19 mg of2-amino-3-{4-[6-(3-cyclopentyloxy-4-methoxy-benzylamino)-pyrazin-2-yl]-phenyl}-propionicacid, yield 63%. ¹H-NMR (400 MHz, CD₃OD): δ 2.22 (s, 3H), 3.09 (m, 1H),3.25 (m, 1H), 4.18 (t, 1H), 4.40 (s, 2H), 7.07 (d, 2H), 7.14 (m, 3H),7.24 (m, 4H), 7.36 (m, 1H), 7.72 (d, 2H), 7.84 (s, 1H), 8.20 (d, 1H).

6.20. Synthesis of(2S)-2-Amino-3-(4-(6-(2,2,2-trifluoro-1-phenylethoxy)-pyrimidin-4-yl)phenyl)propanoicacid

NaH (60%, 120 mg, 3.0 mmol) was added to a solution of2,2,2-trifluoro-1-phenyl-ethanol (350 mg, 2.03 mmol) in 5 ml of THF. Themixture was stirred for 20 minutes at room temperature.4,6-Dichloro-pyrimidine (300 mg, 2.03 mmol) was added and then thereaction mixture was heated at 70° C. for 1 hour. After cooling, the THFwas evaporated to provide a residue, which was dissolved in 15 ml ofEtOAc, and then washed with water, and dried over sodium sulfate. Thesolvent was removed by rotovap to give 550 mg of4-chloro-6-(2,2,2-trifluoro-1-phenyl-ethoxy)-pyrimidine, yield 95%.

An Emrys process vial (2-5 ml) for microwave was charged with4-chloro-6-(2,2,2-trifluoro-1-phenyl-ethoxy)-pyrimidine (30 mg, 0.1μmol), 4-borono-L-phenylalanine (32 mg, 0.16 mmol), 1 ml of acetonitrileand 0.6 ml of water. Aqueous sodium carbonate (0.42 ml, 1M) was added toabove solution followed by 10 mol percent of POPd₂ (dihydrogendi-μ-chlorodichlorobis(di-tert-butylphosphinito-κP)dipalladate. Thereaction vessel was sealed and heated to 120° C. for 30 minutes bymicrowave. After cooling, the reaction mixture was evaporated todryness. The residue was dissolved in 2.5 ml of methanol, and theproduct was purified with Prep-LC to give 4.8 mg of2-amino-3-{4-[6-(2,2,2-trifluoro-1phenyl-ethoxy)-pyrimidin-4-yl]-phenyl}-propionicacid, yield 11%. ¹H-NMR (400 MHz, CD₃OD): δ 3.20 (m, 1H), 3.40 (m, 1H),4.25 (t, 1H), 6.82 (dd, 1H), 7.43 (m, 5H), 7.57 (s, 1H), 7.60 (m, 2H),8.10 (d, 2H), 8.75 (s, 1H).

6.21. Synthesis of(2S)-2-Amino-3-(4-(6-(1-(3,4-difluorophenyl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoicacid

Tetrabutylammonium fluoride (TBAF: 0.1 ml, 1M) in THF was added to asolution of 3,4-difluoro-benzaldehyde (1.42 g, 10 mmol) and(trifluoromethyl)trimethylsilane (1.70 g, 12 mmol) in 10 ml THF at 0° C.The mixture was warmed up to room temperature and stirred for 4 hours.The reaction mixture was treated with 12 ml of 1M HCl and stirredovernight. The product was extracted with dichloromethane (3×20 ml), theorganic layer was combined and passed through a pad of silica gel. Theorganic solvent was evaporated to give 1.9 g of1-(3,4-difluoro-phenyl)-2,2,2-trifluoro-ethanol, yield 90%.

NaH (80 mg, 60%, 3.0 mmol) was added to a solution of1-(3,4-Difluoro-phenyl)-2,2,2-trifluoro-ethanol (212 mg, 1 mmol) in 5 mlof THF, the mixture was stirred for 20 minutes at room temperature.4,6-Dichloro-pyrimidine (149 mg, 1 mmol) was added and then the reactionmixture was heated at 70° C. for 1 hour. After cooling, THF wasevaporated. The residue was dissolved in 15 ml of EtOAc, and then washedwith water, dried over sodium sulfate. The solvent was removed byrotovap to give 230 mg of4-chloro-6-[1-(3,4-difluoro-phenyl)-2,2,2-trifluoro-ethoxy]-pyrimidine,yield 70%.

An Emrys process vial (2-5 ml) for microwave was charged with4-chloro-6-[1-(3,4-difluoro-phenyl)-2,2,2-trifluoro-ethoxy]-pyrimidine(33 mg, 0.1 mmol), 4-borono-L-phenylalanine (31 mg, 0.15 mmol), 1 ml ofacetonitrile and 0.7 ml of water. Aqueous sodium carbonate (0.3 ml, 1M)was added to above solution followed by 5 mol % ofdichlorobis(triphenylphosphine)-palladium(II). The reaction vessel wassealed and heated to 150° C. for 5 minutes by microwave. After cooling,the reaction mixture was evaporated to dryness. The residue wasdissolved in 2.5 ml of methanol, then purified with Prep-LC to give 10mg of2-amino-3-(4-{6-[1-(3,4-difluoro-phenyl)-2,2,2-trifluoro-ethoxy]-pyridin-4-yl}-phenyl)-propionicacid, yield 21%. ¹H-NMR (400 MHz, CD₃OD): δ 3.11 (m, 1H), 3.27 (m, 1H),4.19 (dd, 1H), 6.78 (q, 1H), 7.26 (m, 2H), 7.35 (d, 3H), 7.49 (m, 2H),8.02 (d, 2H), 8.66 (s, 1H).

6.22. Synthesis of(S)-2-Amino-3-(4-(5-(3-(cyclopentyloxy)-4-methoxybenzylamino)-pyrazin-2-yl)phenyl)propanoicacid

A mixture of 3-cyclopentyloxy-4-methoxy-benzaldehyde (417 mg, 1.895mmol), 2-amino-5-bromopyrazine (300 mg, 1.724 mmol), sodiumtriacetoxyborohydride (1.5 eq) and glacial acetic acid (3 eq) indichloromethane (10 ml) was stirred at room temperature overnight. Thenthe reaction mixture was diluted with ethyl acetate, and washed withwater. The organic layer was dried over MgSO₄ and filtered. The filtratewas concentrated to give the crude product, which was purified by ISCO(SiO₂ flash column chromatography) (Hexane/ethyl acetate=100/0 to 3/2)to give about 400 mg of6-bromo-pyrazin-2-yl)-(3-cyclopentyloxy-4-methoxy-benzyl)-amine. Yield:61%.

To a 5 ml microwave vial, the above6-bromo-pyrazin-2-yl)-(3-cyclopentyloxy-4-methoxy-benzyl)-amine (50 mg,0.132 mmol), 4-borono-L-phenylalanine (30 mg, 0.144 mmol), Na₂CO₃ (31mg, 0.288 mmol), acetonitrile (2 ml) and water (2 ml). Dichlorobis(triphenylphosphine)-palladium (5 mg, 0.007 mmol) was added. The vialwas capped and stirred at 150° C. for 5 minutes under microwaveradiation. The reaction mixture was cooled, filtered through a syringefilter and then separated by a reverse phase preparative-HPLC usingYMC-Pack ODS 100×30 mm ID column (MeOH/H₂O/TFA solvent system). The purefractions were concentrated in vacuum. The product was then suspended in5 ml of water, frozen and lyophilized to give the title compound as atrifluoro salt (12 mg, 20%). ¹H NMR (CD₃OD) δ 8.41 (s, 1H), 7.99 (s,1H), 7.83 (d, J=9.0 Hz, 2H), 7.37 (d, J=6.0 Hz, 2H), 6.90-6.95 (m, 3H),4.78 (m, 1H), 4.50 (s, 2H), 4.22-4.26 (m, 1H), 3.79 (s, 3H), 3.12-3.39(m, 2H), 1.80-1.81 (m, 6H), 1.60 (m, 2H). M+1=463.

6.23. Synthesis of(S)-2-Amino-3-(4-(5-((3-(cyclopentyloxy)-4-methoxybenzyl)-(methyl)amino)pyrazin-2-yl)phenyl)propanoicacid

To a solution of(6-bromo-pyrazin-2-yl)-(3-cyclopentyloxy-4-methoxy-benzyl)-amine (70 mg,0.185 mmol) in acetonitrile (10 ml) was added formaldehyde (18.5 mmol)and sodium cyanoborohydride (17 mg, 0.278 mmol). Then, concentratedaqueous HCl was added dropwise until the pH 2. The mixture was stirredfor about 6 hours at room temperature. It was then diluted with ethylacetate, washed with water (3×5 ml), dried over MgSO₄. The solvent wasremoved by vacuum to give 70 mg of crude product5-(bromo-pyrazin-2-yl)-(3-cyclopentyloxy-4-methoxy-benzyl)-methyl-amine(95% crude yield), which was used in the next step without furtherpurification.

The5-(bromo-pyrazin-2-yl)-(3-cyclopentyloxy-4-methoxy-benzyl)-methyl-amine(37 mg, 0.094 mmol) was subjected to a Suzuki coupling reaction asdescribed above to afford 6 mg of the title compound. Yield: 13%. ¹H NMR(CD₃OD) δ 8.59 (s, 1H), 8.12 (s, 1H), 7.85 (d, 2H), 7.39 (d, 2H),6.81-6.91 (m, 3H), 4.72 (m, 1H), 4.30 (m, 1H), 3.79 (s, 3H), 3.20-3.40(m, 2H), 3.18 (s, 3H), 3.79 (s, 3H), 1.80 (m, 6H), 1.58 (m, 2H).M+1=477.

6.24. Synthesis of(S)-2-Amino-3-(4-(5-((1,3-dimethyl-1H-pyrazol-4-yl)methylamino)pyrazin-2-yl)phenyl)propanoicacid

A mixture of 1,3-dimethyl-1H-pyrazole-4-carbaldehyde (142 mg, 1.145mmol), 2-amino-5-bromopyrazine (200 mg, 1.149 mmol), boranetrimethylamine complex (126 mg, 1.73 mmol) and glacial acetic acid (137mg, 2.29 mmol) in anhydrous methonol (3 ml) was stirred at roomtemperature overnight. The reaction mixture was then diluted with ethylacetate, washed with water, dried over MgSO₄ and filtered. The filtratewas concentrated to give 300 mg of(5-bromo-pyrazin-2-yl)-(1,3-dimethyl-1H-pyrazol-4-ylmethyl)amine ascrude product, which was used for next step reaction without furtherpurification. Crude yield: 93%.

The (5-bromo-pyrazin-2-yl)-(1,3-dimethyl-1H-pyrazol-4-ylmethyl)amine (40mg, 0.142 mmol) was used in the Suzuki coupling reaction described aboveto afford 19 mg of the title compound. Yield: 36.5%. ¹H NMR (CD₃OD) δ8.48 (s, 1H), 8.05 (s, 1H), 7.87 (d, 2H), 7.39 (d, 2H), 6.10 (s, 1H),4.81 (s, 2H), 4.30 (m, 1H), 3.83 (s, 3H), 3.11-3.38 (m, 2H), 2.10 (s,3H). M+1=367.

6.25. Synthesis of(S)-2-Amino-3-(4-(4-amino-6-((S)-1-(naphthalen-2-yl)ethylamino)-1,3,5-triazin-2-yloxy)phenyl)propanoicacid

To a 250 ml flask, R-(+)-1-(2-naphthyl)ethylamine (400 mg, 2.424 mmol),2-amino-4,6-dichloro triazine (373 mg, 2.181 mmol), anhydrous1,4-dioxane (40 ml), and N,N-diisopropylethylamine (1 ml, 5.732 mmol)were added and heated to mild reflux for about 4 hours. The reaction wasmonitored carefully in order to avoid the formation of the disubstitutedproduct. (It was observed that the longer the reaction, the moredisubstituted product is formed). After 4 hours, the reaction mixturewas cooled and the solvent was removed under reduced pressure. Water wasadded to the residue, and the solution was sonicated for 2-3 minutes.The solvent was then filtered, washed with water and dried to give 540mg (83% crude yield) of the mono-chloride,6-chloro-N-(1-naphthalen-2yl-ethyl)-[1,3,5]triazine-2,2-diamine, whichwas used for the next step reaction without further purification.

A mixture of6-chloro-N-(1-naphthalen-2yl-ethyl)-[1,3,5]triazine-2,2-diamine (90 mg,0.300 mmol), 2-tert-butoxycarbonylamino-3-(4-hydroxy-phenyl)-propionicacid tert-butyl ester (102 mg, 0.303 mmol) and potassium carbonate (82mg, 0.594 mmol) in isopropanol (8 ml) was refluxed over night. Thesolvent was removed under reduced pressure and the residue was suspendedin ethyl acetate. The solid was filtered and washed with ethyl acetate.The filtrate was concentrated and then redissolved in a mixture ofmethanol/water (90:10) and purified by a preparative-LC using a SunfireC18 OBD 100×30 mm ID column (MeOH/H₂O/TFA solvent system). The purefractions were combined and concentrated to give 50 mg of pure product,3-{4-[4-amino-6-(1-naphthalen-2-yl-ethylamino)-[1,3,5]triazin-2yloxy]-phenyl}2-tert-butoxycarbonylamino-propionicacid tert-butyl ester, (28% yield).

The above product (50 mg, 0.083 mmol) was dissolved in trifluoro aceticacid/dichloromethane (8 ml/2 ml) and stirred at room temperature overnight. The solvent was removed under reduced pressure. The residue wasthen redissolved in a mixture of methanol/water (90:10) and purified bya preparative-LC using a Sunfire C18 OBD 100×30 mm ID column(MeOH/H₂O/TFA solvent system). The pure fractions were combined andconcentrated under reduced pressure to afford about 4 ml, which wasfrozen and lyophilized to give 4 mg of the title compound as a TFA salt(11% yield). ¹H NMR (CD₃OD) δ 7.37-7.81 (m, 8H), 7.19 (m, 2H), 6.98 (m,1H), 5.37 (m, 1H), 4.19 (m, 1H), 3.17-3.38 (m, 2H), 1.56 (m, 3H).M+1=445.

6.26. Synthesis of(S)-2-Amino-3-(4-(4-amino-6-((R)-1-(biphenyl-2-yl)-2,2,2-trifluoroethoxy)-1,3,5-triazin-2-yl)phenyl)propanoicacid

A mixture of 1-biphenyl-2-yl-2,2,2-trifluoro-ethanone (300 mg, 1.2mmol), borane tetrahydrofuran complexes (1.2 ml, 1M in THF, 1.2 mmol)and S-2-methyl-CBS-oxazaborolidine (0.24 ml, 1M in toluene, 0.24 mmol)in THF (8 ml) was stirred at room temperature over night. Several dropsof concentrated HCl were added and the mixture was stirred for 30minutes. The product was purified by SiO₂ chromatography (hexane/ethylacetate=100/0 to 3/1) to give 290 mg of1-biphenyl-2-yl-2,2,2-trifluoro-ethanol (96% yield).

The above alcohol (290 mg, 1.151 mmol) was dissolved in anhydrous THF(10 ml). Sodium hydride (55 mg, 1.375 mmol) was added all at once, andthe mixture was stirred at room temperature for 30 minutes. The solutionwas then transferred into a flask that contained a suspension of2-amino-4,6-dichloro-triazine (190 mg, 1.152 mmol) in THF (20 ml). Themixture was stirred at room temperature overnight. Water was added andthe mixture was then diluted with ethyl acetate. The organic layer waswashed with water, dried over MgSO₄ and then concentrated to give 400 mgof crude product2-amino-4-(1-biphenyl-2-yl-2,2,2-trifluoro-ethoxy-6-chloro-triazine.

The 2-amino-4-(1-biphenyl-2-yl-2,2,2-trifluoro-ethoxy-6-chloro-triazine(40 mg, 0.105 mmol) was subjected to the same Suzuki coupling reactionas described above to afford 5 mg of the title compound. Yield: 9.4%. ¹HNMR (CD₃OD) δ 8.18 (d, 2H), 7.86 (m, 1H), 7.40-7.52 (m, 9H), 7.32 (m,1H), 7.07 (m, 1H), 4.32 (m, 1H), 3.22-3.41 (m, 2H). M+1=510.

6.27. Synthesis of(2S)-2-Amino-3-(4-(4-amino-6-(1-(6,8-difluoronaphthalen-2-yl)ethylamino)-1,3,5-triazin-2-yl)phenyl)propanoicacid

In a three-neck flask, copper iodine (CuI) (299 mg, 1.515 mmol) andlithium chloride (LiCl) (145 mg, 3.452 mmol) were added under nitrogento anhydrous THF (60 ml). The mixture was stirred at room temperatureuntil a pale yellow solution was obtained. After cooling to 0° C.,methyl vinyl ketone and chlorotrimethylsilane were added, and themixture was stirred until an orange color was observed (˜20 min). Aftercooling to about −40° C., a solution of 3,5-difluorophenylmagnesiumbromide (27.65 ml, 13.8 mmol) in THF (0.5M) was slowly added. Thereaction mixture was stirred at about −40° C. for 0.5 hours, then thecold bath was removed and the temperature was allowed to rise slowly toroom temperature. The solvent was evaporated and the residue wasextracted with hexane (4×20 ml). The collected extractions were washedwith cold 10% aqueous NaHCO₃ and dried over Na₂SO₄. The solvent wasevaporated at reduced pressure to afford3,5-difluorophenyl-1-trimethylsilyloxyalkene (2.03 g, 7.929 mmol, 57%crude yield), which was used in the successive reaction without furtherpurification.

Powered calcium carbonate (3.806 g, 38.06 mmol) and ethyl vinyl ether(2.184 g, 30.329 mmol) were added to a solution of ceric ammoniumnitrate (10.430 g, 19.033 mmol) in methanol (40 ml) under nitrogenatmosphere. To the resulting suspension was added a solution of abovemade 3,5-difluorophenyl-1-trimethylsilyloxyalkene (2.03 g, 7.929 mmol)in ethyl vinyl (6 ml, 4.518 g, 62.75 mmol) dropwise under vigorousstirring, and the mixture was stirred at room temperature overnight. Thesolid was filtered through a celite layer, and the filtrate wasconcentrated to one-fourth of its initial volume. The resulting thickmixture was slowly poured, under vigorous stirring, into 1:1 v/v diethylether-10% aqueous NaHCO₃. The precipitate was filtered off, the etherealsolution was separated, and the solvent was evaporated at reducedpressure to give clear liquid. The solution of resulting liquid (amixture of acyclic and cyclic acetates) in methanol (4 ml) was addeddropwise to a suspension of dichlorodicyanobenzoquinone (1.77 g, 7.797mmol) in 80% aqueous sulfuric acid at 0° C. After the addition wascomplete, the ice bath was removed and stirring was continued for 30minutes. The mixture was poured into ice water; and the resulting brownprecipitate was filtered and dissolved in acetone. Silica gel was addedto make a plug, and the crude product was purified by chromatography(hexane/ethyl acetate=100/0 to 3/1) to give 760 mg of1-(5,7-difluoro-naphthalen-2-yl)-ethanone (48% in two-step yield) as alight yellow solid.

The above ketone (760 mg, 3.689 mmol) was dissolved in methanol (40 ml).Then, ammonium acetate (2.841 g, 36.896 mmol), sodium cyanoborohydride(232 mg, 3.389 mmol) and molecular sieves (3 Å, 7.6 g) were added. Themixture was stirred at room temperature for two days. The solid wasfiltered and the filtrate was concentrated. The residue was dissolved inwater and concentrated aqueous HCl was added dropwise until the pH≈2.The mixture was then extracted with ethyl acetate to remove theunfinished ketone and other by-products. The water layer was basified topH 10 with aqueous sodium hydroxide (1M), and was extracted withdichloromethane and the organic layers were combined, dried overmagnesium sulfate and concentrated to afford 290 mg of1-(5,7-difluoro-naphthalen-2-yl)-ethylamine (38% yield).

The fresh made amine (290 mg, 1.401 mmol) was added directly to asuspension of 2-amino-4,6-dichloro triazine (277 mg, 1.678 mmol) inanhydrous 1,4-dioxane (60 ml), and followed by addition ofN,N-diisopropylethylamine (1 ml, 5.732 mmol). The mixture was heated tomild reflux for about 3 hours. The reaction mixture was then cooled, andthe solvent was removed under reduced pressure. To the residue was addedwater and the mixture was sonicated for 2-3 minutes. The resulting solidwas filtered and washed with water and dried to give 395 mg (60% crudeyield) of6-chloro-N-[1-(6,8-difluoronaphthalen-2-yl-ethyl]-[1,3,5]triazine-2,4-diamine,which was used for the next step reaction directly without furtherpurification.

The above made mono-chloride (48 mg, 0.144 mmol) was subjected to thesame Suzuki coupling reaction as described above to afford 12 mg of thetitle product. Yield: 17.9%. ¹H NMR (CD₃OD) δ 8.14-8.22 (m, 2H), 8.05(m, 1H), 7.92 (m, 1H), 7.63 (m, 1H), 7.32-7.51 (m, 3H), 7.11 (m, 1H),5.48 (m, 1H), 4.13 (m, 1H), 3.13-3.41 (m, 2H), 1.66 (d, 3H). M+1=465.

6.28. Synthesis of(2S)-2-Amino-3-(4-(4-amino-6-(2,2,2-trifluoro-1-(3′-methylbiphenyl-2-yl)ethoxy)-1,3,5-triazin-2-yl)phenyl)propanoicacid

To a mixture of 3′-methyl-1-biphenyl-2-carbaldehyde (500 mg, 2.55 mmol)and trifluoromethyl trimethylsilane (435 mg, 3.061 mmol) in THF (3 ml)was added tetrabutyl ammonium fluoride (13 mg, 0.05 mmol) at 0° C. Thetemperature was allowed to warm to room temperature. The mixture wasstirred for 5 hours at room temperature, then diluted with ethylacetate, washed with water and brine and dried by MgSO₄. The solvent wasremoved under reduced pressure to give 660 mg (97% crude yield) of2,2,2-trifluoro-1-(3′-methyl-biphenyl-2-yl)-ethanol as crude product,which was used for next step without further purification.

The above-made alcohol (660 mg, 2.481 mmol) was dissolved in anhydrous1,4-dioxane (10 ml). Sodium hydride (119 mg, 60% in mineral oil, 2.975mmol) was added all at once and the mixture was stirred at roomtemperature for 30 minutes. The solution was transferred into a flaskcontaining a suspension of 2-amino-4,6-dichloro-triazine (491 mg, 2.976mmol) in 1,4-dioxane (70 ml). The mixture was stirred at roomtemperature for 6 hours. The solvent was removed, and the residue wassuspended in ethyl acetate, which was washed with water, dried overMgSO₄ and then concentrated to give 790 mg of crude product, whichcontained about 57% of the desired product2-amino-4-(1-(3′-methyl-biphenyl-2-yl-2,2,2-trifluoro-ethoxy-6-chloro-triazineand about 43% byproduct (the bisubstituted product). The crude productwas used without further purification.

The2-amino-4-(1-(3′-methyl-biphenyl-2-yl-2,2,2-trifluoro-ethoxy-6-chloro-triazine(98 mg, 57% purity, 0.142 mmol) was used to run the same Suzuki couplingreaction as described above to afford 9 mg of the title compound. Yield:12.0%. ¹H NMR (CD₃OD) δ 8.09 (m, 2H), 7.85 (m, 1H), 7.50 (m, 2H),7.28-7.43 (m, 5H), 7.17-7.26 (m, 2H), 7.18 (m, 1H), 3.85 (m, 1H),3.08-3.44 (m, 2H), 2.33 (s, 3H). M+1=524.

6.29. Synthesis of(S)-2-Amino-3-(4-(5-(3,4-dimethoxyphenylcarbamoyl)-pyrazin-2-yl)phenyl)propanoicacid

To a mixture of 3,4-dimethoxy phenylamine (0.306 g, 2 mmol) andtriethylamine (0.557 ml, 4 mmol) in dichloromethane (20 ml) was added5-chloro-pyrazine-2-carbonyl chloride (0.354 g, 2 mmol) at 0-5° C. Themixture was allowed to stir at room temperature for 3 hours. The mixturewas diluted with methylene chloride (20 ml), washed with saturatedNaHCO₃ (20 ml), brine (20 ml), dried (anhyd. Na₂SO₄) and concentrated toget 0.42 g of crude 5-chloro-pyrazine-2 carboxylic acid(3,4-dimethoxy-phenyl)-amide, which was directly used in the nextreaction.

5-Chloro-pyrazine-2 carboxylic acid (3,4-dimethoxy-phenyl)-amide (0.18g, 0.61 mmol), L-p-borono phenylalanine (0.146 g, 0.70 mmol), CH₃CN (2.5ml), H₂O (2.5 ml), Na₂CO₃ (0.129 g, 1.22 mmol) were combined in amicrowave vial. The mixture was sealed and kept at 150° C. for 5minutes. The mixture was filtered and concentrated. The residue wasdissolved in methanol/water (1:1) and purified by preparative HPLC,using MeOH/H₂O/TFA as solvent system to afford2-amino-3-{4-[5-(3,4-dimethoxy-phenylcarbornyl)-pyrazin-2yl]-phenyl}-propionicacid as a TFA salt (HPLC: Method A, Retention time=2.846 min, LCMS M+1423). ¹H NMR (400 MHz, DMSO-d₆) δ 3.10-3.30 (m, 2H), 3.72 (d, 6H), 4.05(m, 1H), 7.42-7.62 (m, 4H), 8.22 (m, 3H), 9.30 (m, 2H).

6.30. Synthesis of(S)-2-Amino-3-(4-(2-amino-6-(4-(2-(trifluoromethyl)phenyl)-piperidin-1-yl)pyrimidin-4-yl)phenyl)propanoicacid

2-Amino 4,6-dichloro pyrimidine (0.164 g, 1 mmol),4-(2-trifluoromethyl-phenyl)-piperidine hydrochloride (0.266 g, 1 mmol),and cesium carbonate (0.684 g, 2.1 mmol) were dissolved in a mixture of1,4-dioxane (5 ml) and H₂O (5 ml) in a 20 ml microwave vial. The mixturewas stirred at 210° C. for 20 minutes in a microwave reactor. Solventwas removed and the residue was dissolved in 5% methanol in CH₂Cl₂ (20ml), dried over Na₂SO₄ and concentrated to get the crude intermediate,4-chloro-6-[4-(2-trifluoromethyl-phenyl)-piperidin-1-yl]-pyrimidin-2-ylamine(0.42 g) which was directly used in the following step.

The crude intermediate (0.42 g), L-p-borono-phenylalanine (0.209 g, 1mmol), sodium carbonate (0.210 g, 2 mmol), and dichlorobis(triphenylphosphine)-palladium(II) (35 mg, 0.05 mmol) were dissolved ina mixture of MeCN (2.5 ml) and H₂O (2.5 ml) in a 10 ml microwave vial.The vial was sealed and stirred in a microwave reactor at 150° C. for 6minutes. The mixture was filtered, and the filtrate was concentrated.The residue was dissolved in MeOH and H₂O (1:1) and purified bypreparative HPLC using MeOH/H₂O/TFA as the solvent system to afford2-amino-3-(4-{4-(2-trifluoromethyl-phenyl)-piperidine-1-yl]-pyrimidin-4yl}-phenyl)-propionicacid as a TFA salt. HPLC: Method A, Retention time=3.203 min. LCMS M+1486. ¹H NMR (400 MHz, CD₃OD) δ 1.80-2.20 (m, 5H), 3.0-3.16 (m, 2H),3.22-3.42 (m, 2H), 4.22 (t, 1H), 4.42-4.54 (m, 1H), 5.22-5.34 (m, 1H),6.80 (s, 1H), 7.40 (t, 1H), 7.50-7.60 (m, 4H), 7.68 (d, 1H), 7.82 (d,2H).

6.31. Synthesis of(S)-2-Amino-3-(4-(2-amino-6-((R)-1-(naphthalen-2-yl)ethylamino)pyrimidin-4-yl)phenyl)propanoicacid

2-Amino 4,6-dichloro pyrimidine (0.164 g, 1 mmol),(R)-(+)-1-(2-naphthyl)-ethylamine (0.171 g, 1 mmol), and cesiumcarbonate (0.358 g, 1.1 mmol) were dissolved in a mixture of 1,4-dioxane(4 ml) and H₂O (4 ml) in a 20 ml microwave vial. The vial was sealed andstirred at 210° C. for 20 minutes in a microwave reactor. Solvent wasremoved and the residue was dissolved in CH₂Cl₂ (50 ml), washed withwater (20 ml), brine (20 ml), dried (Na₂SO₄) and concentrated to affordthe crude intermediate,6-chloro-N-4-(naphthalene-2yl-ethyl)-pyrimidine-2,4-diamine (0.270 g)which was directly used in the following step.

The crude intermediate (0.27 g), L-p-borono-phenylalanine (0.210 g, 1mmol), sodium carbonate (0.210 g, 2 mmol), anddichlorobis(triphenylphosphine)-palladium(II) (25 mg, 0.036 mmol) weredissolved in a mixture of MeCN (2.5 ml) and H₂O (2.5 ml) in a microwavevial. The vial was sealed and stirred in the microwave reactor at 150°C. for 6 minutes. The mixture was filtered and the filtrate wasconcentrated. The residue was dissolved in MeOH and H₂O (1:1) andpurified by preparative HPLC using MeOH/H₂O/TFA as the solvent system toafford 2amino-3-{4-[2-amino-6-(1-naphthalen-2yl-ethylamino)-pyrimidin-4-yl]-phenyl}-propionicacid as a TFA salt. HPLC: Method A, Retention time=3.276 min. LCMS M+1428. ¹H NMR (400 MHz, CD₃OD) δ 1.68 (d, 3H), 3.22-3.40 (m, 2H), 4.30 (t,1H), 5.60 (q, 1H), 6.42 (s, 1H), 7.42-7.54 (m, 5H), 7.72 (m, 2H),7.82-7.84 (m, 4H).

6.32. Synthesis of(S)-2-Amino-3-(4-(2-amino-6-(methyl((R)-1-(naphthalen-2-yl)ethyl)amino)pyrimidin-4-yl)phenyl)propanoicacid

2-Amino 4,6-dichloro pyrimidine (0.327 g, 2 mmol),methyl-(1-naphthalen-2yl-ethyl)-amine (0.360 g, 2 mmol), and cesiumcarbonate (0.717 g, 2.2 mmol) were dissolved in a mixture of 1,4-dioxane(7.5 ml) and H₂O (7.5 ml) in a 20 ml microwave vial. The vial was sealedand stirred at 210° C. for 20 minutes in a microwave reactor. Solventwas removed and the residue was dissolved in CH₂Cl₂ (50 ml), washed withwater (20 ml), brine (20 ml) dried (Na₂SO₄) and concentrated to get thecrude intermediate,6-chloro-N-4-methyl-N-4-(1-napthalen-2-yl-ethyl)-pyrimidine-2,4-diamine(0.600 g), which was directly used in the following step.

The crude intermediate (0.30 g), L-p-borono-phenylalanine (0.210 g, 1mmol), sodium carbonate (0.210 g, 2 mmol), anddichlorobis(triphenylphosphine)-palladium(II) (25 mg, 0.036 mmol) weredissolved in a mixture of MeCN (2.5 ml) and H₂O (2.5 ml) in a microwavevial. The vial was sealed and stirred in the microwave reactor at 150°C. for 6 minutes. The mixture was filtered and the filtrate wasconcentrated. The residue was dissolved in MeOH and H₂O (1:1) andpurified by preparative HPLC using MeOH/H₂O/TFA as the solvent system toafford2-amino-3-(4-{2-amino-6-[methyl-(1-naphthalen-2yl-ethyl)amino]-pyrimidin-4yl}-phenyl)-propionicacid as a TFA salt (HPLC: Method C, Retention time=2.945 min, LCMS M+1442) ¹H NMR (400 MHz, CD₃OD) δ 1.70 (m, 3H), 2.92 (s, 3H), 3.22-3.42 (m,2H), 4.28 (m, 1H), 6.60 (s, 1H), 6.72 (m, 1H), 7.40-7.92 (m, 11H).

6.33. Synthesis of(S)-2-Amino-3-(4-(2-amino-6-((S)-2,2,2-trifluoro-1-(6-methoxynaphthalen-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoicacid

2-Amino 4,6-dichloro pyrimidine (0.096 g, 0.6 mmol),2,2,2-trifluoro-1-(6-methoxy-naphthalen-2-yl)-ethanol (0.140 g, 0.55mmol), and NaH (96 mg, 0.60 mmol) were added to anhydrous dioxane (20ml) under a nitrogen atmosphere. The reaction was stirred at 80° C. for12 hours, cooled to room temperature, and quenched with water (0.2 ml).The reaction mixture was concentrated, and the residue dissolved inCH₂Cl₂ (50 ml), washed with water (20 ml), brine (20 ml) dried (Na₂SO₄)and concentrated to afford the crude intermediate,4-chloro-6-[2,2,2-trifluoro-1-(6-methoxy-naphthalene-2-yl)-ethoxy]-pyrimidin-2-ylamine(0.22 g) which was directly used in the following step.

The crude intermediate (0.22 g), L-p-borono-phenylalanine (0.126 g, 0.6mmol), sodium carbonate (0.126 g, 1.2 mmol), anddichlorobis(triphenylphosphine)-palladium(II) (15 mg, 0.021 mmol) weredissolved in a mixture of MeCN (2.0 ml) and H₂O (2.0 ml) in a microwavevial. The vial was sealed and stirred in the microwave reactor at 150°C. for 6 minutes. The mixture was filtered and the filtrate wasconcentrated. The residue was dissolved in MeOH and H₂O (1:1) andpurified by preparative HPLC using MeOH/H₂O/TFA as the solvent system toafford2-amino-3-(4-{2-amino-6-[2,2,2-trifluoro-1-(6-methoxy-naphthalen-2-yl)-ethoxy]-pyrimidin-4-yl}-phenyl)-propionicacid as a TFA salt (HPLC: Method C, Retention time=3.190 min. LCMS M+1513. ¹H NMR (400 MHz, CD₃OD) δ 3.22-3.42 (m, 2H), 3.86 (s, 3H), 4.32(1H), 6.88 (m, 1H), 6.92 (1H), 7.20 (dd, 1H), 7.26 (s, 1H), 7.50 (d,2H), 7.63 (d, 1H), 7.80-7.90 (m, 4H), 8.05 (s, 1H).

6.34. Synthesis of(S)-2-Amino-3-(4-(5-(biphenyl-4-ylmethylamino)pyrazin-2-yl)phenyl)propanoicacid

4-Phenylbenzaldehyde (0.3 g, 1.65 mmol) and 2-amino-5-bromopyrazine(0.24 g, 1.37 mmol) were treated with Na(OAc)₃BH (0.44 g, 2.06 mmol) indichloroethane (7.0 mls) and acetic acid (0.25 mls) for 18 hours at roomtemperature. The mixture was diluted with dichloromethane, washed with1.0 N NaOH, washed with brine, dried over MgSO₄, and concentrated.Chromatography (SiO₂, EtOAc:Hex, 1:1) gave 0.18 g ofN-(biphenyl-4-ylmethyl)-5-bromopyrazin-2-amine.

N-(biphenyl-4-ylmethyl)-5-bromopyrazin-2-amine (60 mg, 0.176 mmol),L-p-boronophenylalanine (37 mg, 0.176 mmol), palladiumtriphenylphosphinedichloride (3.6 mg, 0.0052 mmol), Na₂CO₃ (37 mg, 0.353 mmol),acetonitrile (1.25 mls) and water (1.25 mls) were heated in a microwavereactor at 150° C. for 5 minutes. The mixture was concentrated,dissolved in 1.0 N HCl, washed twice with ether, concentrated andpurified by preprative HPLC to give 41 mgs of the title compound.M+1=425; ¹H NMR (CD₃OD) δ 8.42 (s, 1H), 8.05 (s, 1H), 7.92 (d, 2H), 7.58(d, 4H), 7.40 (m, 7H), 4.60 (s, 2H), 4.25 (m, 1H), 3.40 (m, 1H), 3.20(m, 1H).

6.35. Synthesis of(S)-2-Amino-3-(4-(5-(naphthalen-2-ylmethylamino)pyrazin-2-yl)phenyl)propanoicacid

2-Napthaldehyde (0.6 g, 3.84 mmol) and 2-amino-5-bromopyrazine (0.56 g,3.201 mmol) were treated with Na(OAc)₃BH (1.02 g, 4.802 mmol) indichloroethane (15.0 mls) and acetic acid (0.5 mls) for 18 hours at roomtemperature. The mixture was diluted with dichloromethane, washed with1.0 N NaOH, washed with brine, dried over MgSO₄, and concentrated.Chromatography (SiO₂, EtOAc:Hex, 1:1) gave 0.49 g5-bromo-N-(naphthalen-2-ylmethyl)pyrazin-2-amine.

5-Bromo-N-(naphthalen-2-ylmethyl)pyrazin-2-amine (0.2 g, 0.637 mmol),L-p-boronophenylalanine (0.13 g, 0.637 mmol),palladiumtriphenylphosphine dichloride (13 mg, 0.019 mmol), Na₂CO₃ (0.13g, 1.27 mmol), acetonitrile (5 mls) and water (5 mls) were heated in amicrowave reactor at 150° C. for 5 minutes. The mixture wasconcentrated, dissolved in 1.0 N HCl, washed twice with ether,concentrated, dissolved in methanol, filtered and concentrated to yield0.12 g of the captioned compound. M+1=399; ¹H NMR (CD₃OD) δ 8.51 (s,1H), 8.37 (s, 1H), 7.90 (m, 6H), 7.50 (m, 5H), 4.85 (s, 2H), 4.30 (t,1H), 3.38 (m, 1H), 3.22 (m, 1H).

6.36. Synthesis of(S)-2-(Tert-butoxycarbonylamino)-3-(4-(5-(naphthalen-2-ylmethylamino)pyrazin-2-yl)phenyl)propanoicacid

(S)-2-Amino-3-(4-(5-(naphthalen-2-ylmethylamino)pyrazin-2-yl)phenyl)propanoicacid (0.15 g, 0.345 mmol) was treated with triethylamine (87 mg, 0.862mmol), and boc-anhydride (84 mg, 0.379) in dioxane (3 ml) and H₂O (3 ml)at 0° C. The mixture was warmed to room temperature and stirredovernight. The mixture was concentrated, and partitioned between EtOAcand H₂O. The aqueous phase was acidified to pH=1 with 1.0 N HCl andextracted with EtOAc. The organics were combined, washed with brine,dried over MgSO₄, and concentrated to yield 48 mg of the captionedcompound.

6.37. Synthesis of (S)-2-Morpholinoethyl2-amino-3-(4-(5-(naphthalen-2-ylmethylamino)pyrazin-2-yl)phenyl)propanoate

(S)-2-(Tert-butoxycarbonylamino)-3-(4-(5-(naphthalen-2-ylmethylamino)pyrazin-2-yl)phenyl)propanoicacid (48 mg, 0.090 mmol), 4-(2-hydroxyethyl)morpholine (12 mg, 0.090mmol), triethylamine (18 mg, 0.180 mmol), andbenzotriazole-1-yloxytris(dimethylamino)-phosphonium hexafluorophosphate(BOP, 18 mg, 0.090 mmol), in dichloromethane (3.0 ml) were stirred atroom temperature for 5 hours. Additional triethylamine (18 mg, 0.180mmol) and BOP (18 mg, 0.090 mmol) were added, and the mixture wasstirred overnight. The mixture was concentrated and purified via prepHPLC to give 2 mg of the captioned compound.

6.38. Synthesis of(2S)-2-Amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3′-fluorobiphenyl-4-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoicacid

To 4′-bromo-2,2,2-trifluoroacetophenone (5.0 g, 19.76 mmol) in THF (50mls) at 0° C. was added NaBH₄ (1.5 g, 39.52 mmol). The mixture waswarmed to room temperature and stirred for 1 hour. The reaction wascomplete by TLC (CH₂Cl₂). The mixture was quenched with H₂O, rotaryevaporated to remove most of the THF, and extracted 2 times with CH₂Cl₂.The organics were combined, washed with brine, concentrated to a smallvolume and filtered through a plug of silica gel. The silica was washedwith CH₂Cl₂ to elute the product, and the resulting solution wasconcentrated to give 4.65 g of 1-(4-bromophenyl)-2,2,2-trifluoroethanol.Yield 92%.

To Pd(PPh₃)₄ (2.1 g, 1.823 mmol) was added 3-fluorophenylmagnesiumbromide (55 mls, 1.0 M in THF, 55 mmol) at 0° C. over 15 minutes. Theice bath was removed and the mixture was stirred for 30 minutes.1-(4-Bromophenyl)-2,2,2-trifluoroethanol (4.65 g, 18.23 mmol) in THF (50mls) was added over 10 minutes. The mixture was heated to reflux for 3hours and was shown complete by LC (Sunfire column, TFA). The mixturewas cooled, quenched with H₂O, rotary evaporated to remove most of theTHF, and extracted 3 times with CH₂Cl₂. The organics were combinedwashed with brine, dried over MgSO₄, and concentrated. Chromatography(SiO₂, CH₂Cl₂) gave 4.64 g of2,2,2-trifluoro-1-(3′-fluorobiphenyl-4-yl)ethanol. Yield 94%.

To 2,2,2-trifluoro-1-(3′-fluorobiphenyl-4-yl)ethanol (1.4 g, 5.18 mmol)in THF (50 mls) at 0° C. was added NaH (60% in mineral oil, 0.31 g, 7.77mmol). The ice bath was removed and the mixture was stirred for 30minutes. 2-Amino-4,6-dichloropyrimidine (1.0 g, 6.22 mmol) in THF (25mls) was added at once. The mixture was heated to 50° C. for 5 hours.The reaction was complete by LCMS (Sunfire, TFA). The mixture wascooled, quenched with brine, and extracted 3 times with CH₂Cl₂. Theorganics were combined, washed with brine, dried over MgSO₄, andconcentrated. Chromatography (SiO₂, CH₂Cl₂) afforded 1.48 g of4-chloro-6-(2,2,2-trifluoro-1-(3′-fluorobiphenyl-4-yl)ethoxy)pyrimidin-2-amine.Yield 73%.

4-Chloro-6-(2,2,2-trifluoro-1-(3′-fluorobiphenyl-4-yl)ethoxy)pyrimidin-2-amine(0.75 g, 1.89 mmol), L-p-boronophenylalanine (0.47 g, 2.26 mmol),Pd(PPh₃)₂Cl₂ (79 mgs, 0.113 mmol), Na₂CO₃ (0.44 g, 4.15 mmol),acetonitrile (10 mls), and H₂O (10 mls) were combined in a 20 mlmicrowave reactor and heated in the microwave at 150° C. for 7 minutes.The reaction was complete by LCMS (Sunfire, neutral). The mixture wasconcentrated, dissolved in NaOH (20 mls 0.5 N), filtered, extracted withether three times, and cooled to 0° C. At 0° C., 1.0 N HCl was addedslowly until a pH of 6.5 was attained. The mixture was stirred at 0° C.for 30 minutes and the product was filtered, dried in air, treated withexcess 2.0 N HCl in ether, concentrated, then triturated with CH₂Cl₂ togive 1.12 g, 99% (95.5% purity). 385 mgs were purified via prep HPLC(Sunfire, TFA), concentrated, treated with excess 1.0 N HCl (aq.),concentrated to a small volume and lyophilized to afford 240 mgs of thecaptioned compound. M+1=527; ¹H NMR 6 (CD₃OD) 7.86 (d, 2H), 7.64 (s,4H), 7.49 (d, 2H), 7.36 (m, 2H), 7.28 (m, 1H), 7.02 (m, 1H), 6.95 (s,1H), 6.75 (q, 1H), 4.26 (t, 1H), 3.32 (m, 1H), 3.21 (m, 1H).

6.39. Synthesis of(S)-2-Amino-3-(4-(2-amino-6-(benzylthio)pyrimidin-4-yl)phenyl)propanoicacid

Benzylmercaptan (0.14 g, 1.11 mmol) was treated with NaH (60% in mineraloil, 67 mg, 1.66 mmol) in dry THF (15 ml) for 30 minutes.2-Amino-4,6-dichloropyrimidine (0.2 g, 1.22 mmol) was added and themixture was stirred overnight. The mixture was diluted withmethylenechloride, washed with water, then brine, dried over MgSO₄, andconcentrated to give 0.11 g of 4-(benzylthio)-6-chloropyrimidin-2-amine.

4-(Benzylthio)-6-chloropyrimidin-2-amine (0.1 g, 0.397 mmol),L-p-boronophenylalanine (0.1 g, 0.477 mmol), Pd(PPh₃)₂Cl₂ (17 mg, 0.024mmol), Na₂CO₃ (93 mg, 0.874 mmol), MeCN (2.5 ml) and water (2.5 ml) wereheated at 150° C. for 5 minutes in a microwave. The mixture wasconcentrated and purified via prep HPLC to give 0.42 g of the titlecompound. M+1=381; ¹H NMR (CD₃OD) δ 7.8 (d, 2H), 7.37 (t, 4H), 7.23 (m,2H), 7.16 (m, 1H), 6.98 (s, 1H), 4.43 (s, 2H), 4.20 (t, 1H), 3.29 (m,1H), 3.13 (M, 1H).

6.40. Synthesis of(S)-2-Amino-3-(4-(2-amino-6-(naphthalen-2-ylmethylthio)pyrimidin-4-yl)phenyl)propanoicacid

2-Mercaptonapthalene (0.2 g, 1.148) was treated with NaH (60% in Mineraloil, 92 mg, 2.30 mmol) in dry THF (10 ml) for 30 minutes.2-Amino-4,6-dichloropyrimidine (0.21 g, 1.26 mmol) was added and themixture was stirred overnight. The mixture was diluted withmethylenechloride, washed with water, then brine, dried over MgSO₄, andconcentrated to give 0.18 g4-chloro-6-(naphthalen-2-ylmethylthio)pyrimidin-2-amine.

4-Chloro-6-(naphthalen-2-ylmethylthio)pyrimidin-2-amine (0.1 g, 0.331mmol), L-p-boronophenylalanine (83 mg, 0.397 mmol), Pd(PPh₃)₂Cl₂ (14 mg,0.020 mmol), Na₂CO₃ (77 mg, 0.729 mmol), MeCN (2.5 ml) and water (2.5ml) were heated at 150° C. for 5 minutes in a microwave. The mixture wasconcentrated and purified via prep HPLC to give 57 mg of the titlecompound. M+1=431; ¹H NMR (CD₃OD) δ 7.85 (s, 1H), 7.79 (d, 2H), 7.72 (d,3H), 7.46 (dd, 1H), 7.35 (m, 4H), 6.95 (s, 1H), 4.58 (s, 2H), 4.17 (m,1H), 3.26 (m, 1H), 3.11 (m, 1H).

6.41. Synthesis of(2S)-2-Amino-3-(4-(2-amino-6-(1-(3,4-difluorophenyl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoicacid

3,5-Difluorophenyl-trifluoromethyl ketone was treated with NaBH₄ (0.18g, 4.76 mmol) in THF (5 ml) for 2 hours. The mixture was quenched withwater, extracted with methylene chloride (2×). The organics werecombined, filtered through silica gel and concentrated to give 0.46 g of1-(3,4-difluorophenyl)-2,2,2-trifluoroethanol.

1-(3,4-Difluorophenyl)-2,2,2-trifluoroethanol (0.1 g, 0.471 mmol) wastreated with NaH (60% in mineral oil, 38 mg, 0.943 mmol) in dry THF (3ml) for 30 minutes. 2-Amino-4,6-dichloropyrimidine (77 mg, 0.471 mmol)was added and the mixture was stirred at 50° C. for 6 hours. The mixturewas quenched with water and extracted with methylenechloride (2×). Theorganics were combined, washed with water, then brine, dried over MgSO4,and concentrated to give 0.14 g of4-chloro-6-(1-(3,4-difluorophenyl)-2,2,2-trifluoroethoxy)-pyrimidin-2-amine.

4-Chloro-6-(1-(3,4-difluorophenyl)-2,2,2-trifluoroethoxy)pyrimidin-2-amine(0.14 g, 0.421 mmol), L-p-boronophenylalanine (110 mg, 0.505 mmol),Pd(PPh₃)₂Cl₂ (18 mg, 0.025 mmol), Na₂CO₃ (98 mg, 0.926 mmol), MeCN (2.5ml) and water (2.5 ml) were heated at 150° C. for 5 minutes in amicrowave. The mixture was concentrated and purified via prep HPLC togive 74 mg of the title compound. M+1=469; ¹H NMR (CD₃OD) δ 7.83 (d,2H), 7.47 (m, 1H), 7.38 (m, 4H), 7.28 (m, 1H), 4.21 (t, 1H), 3.29 (m,1H), 3.15 (m, 1H).

6.42. Synthesis of(2S)-2-Amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3′-methylbiphenyl-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoicacid

To 4′-bromo-2,2,2-trifluoroacetophenone (5.0 g, 19.76 mmol) in THF (50mls) at 0° C. was added NaBH₄ (1.5 g, 39.52 mmol). The mixture waswarmed to room temperature and stirred for 1 hour. The reaction wascomplete by TLC (CH₂Cl₂). The mixture was quenched with H₂O, rotaryevaporated to remove most of the THF, and extracted 2 times with CH₂Cl₂.The organics were combined, washed with brine, concentrated to a smallvolume and filtered through a plug of silica gel. The silica was washedwith CH₂Cl₂ to elute the product, and the resulting solution wasconcentrated to give 4.65 g of 1-(4-bromophenyl)-2,2,2-trifluoroethanol.Yield: 92%.

1-(4-Bromophenyl)-2,2,2-trifluoroethanol (0.13 g, 0.525 mmol),m-tolylboronic acid (0.1 g, 0.736 mmol), Fibercat (4.28% Pd, 47 mgs,0.0157 mmol Pd), K₂CO₃ (0.22 g, 1.576 mmol), EtOH (3 mls), and H₂O (0.5mls) were combined and heated at 80° C. for 4 hours. The reaction wasshown complete by TLC (CH₂Cl₂). The mixture was cooled, filtered,concentrated, slurried in CH₂Cl₂, and chromatographed over silica gel(CH₂Cl₂) to give 0.1 g of2,2,2-trifluoro-1-(3′-methylbiphenyl-2-yl)ethanol. Yield: 72%.

Alternatively, 1-(4-bromophenyl)-2,2,2-trifluoroethanol (0.98 g, 3.86mmol), m-tolylboronic acid (0.63 g, 4.63 mmol), Pd(PPh₃)₂Cl₂ (0.16 g,0.232 mmol Pd), Na₂CO₃ (0.90 g, 8.49 mmol), AcCN (10 mls), and H₂O (10mls) were combined and heated in the microwave at 150° C. for 10minutes. The reaction was shown complete by TLC (CH₂Cl₂). The mixturewas cooled, concentrated, slurried in CH₂Cl₂, filtered, andchromatographed over silica gel (CH₂Cl₂) to give 0.80 g of2,2,2-trifluoro-1-(3′-methylbiphenyl-2-yl)ethanol. Yield: 79%.

Alternatively, tetrabutylammoniumfluoride (TBAF 1.0 N in THF 13 uL, 3.3mg, 0.013 mmol) was added to a mixture of3-methyl-biphenyl-2-carboxaldehyde (0.25 g, 1.27 mmol) andtrifluoromethytrimethyl silane (0.25 g, 1.53 mmol), in THF (1.5 ml) at0° C. The reaction was warmed to room temperature and stirred for 4hours. HCl (3.0 N, 2.0 ml) was added, and the mixture was stirred for 3hours. The mixture was concentrated, dissolved in methylene chloride,filtered through silica gel, and concentrated to give 0.15 g of2,2,2-trifluoro-1-(3′-methylbiphenyl-2-yl)ethanol.

2,2,2-Trifluoro-1-(3′-methylbiphenyl-2-yl)ethanol (0.15 g, 0.563 mmol)was treated with NaH (60% in mineral oil, 45 mg, 1.12 mmol) in dry THF(5 ml) for 30 minutes. 2-Amino-4,6-dichloropyrimidine (92 mg, 0.5633mmol) was added and the mixture was stirred at 50° C. for 6 hours. Themixture was quenched with water and extracted with methylenechloride(2×). The organics were combined, washed with water, then brine, driedover MgSO4, and concentrated to give 0.16 g of4-chloro-6-(2,2,2-trifluoro-1-(3′-methylbiphenyl-2-yl)ethoxy)pyrimidin-2-amine.

4-Chloro-6-(2,2,2-trifluoro-1-(3′-methylbiphenyl-2-yl)ethoxy)pyrimidin-2-amine(0.16 g, 0.406 mmol), L-p-boronophenylalanine (10 mg, 0.487 mmol),Pd(PPh₃)₂Cl₂ (17 mg, 0.024 mmol), Na₂CO₃ (95 mg, 0.894 mmol), MeCN (2.5ml) and water (2.5 ml) were heated at 150° C. for 5 minutes in amicrowave. The mixture was concentrated and purified via prep HPLC togive 105 mg of the title compound. M+1=523; ¹H NMR (CD₃OD) δ 7.85 (d,2H), 7.70 (d, 1H), 7.44 (m, 4H), 7.31 (t, 1H), 7.21 (m, 2H), 7.10 (m,2H), 6.87 (q, 1H), 6.84 (s, 1H), 4.25 (t, 1H), 3.30 (m, 1H), 3.18 (m,1H).

6.43. Synthesis of(S)-2-Amino-3-(4-(5-(3-(cyclopentyloxy)-4-methoxybenzylamino)pyridin-3-yl)phenyl)propanoicacid

Sodium triacetoxyl-borohydride (245 mg, 1.16 mmol) was added to thesolution of 5-bromo-pyridine-3-amine (100 mg, 0.57 mmol) and3-cyclopentyloxy-4-methoxy-benzaldehyde (127 mg, 0.57 mmol) in 10 ml of1,2-dichloroethtane (DCE), of HOAc (66 μL, 2 eq. 1.16 mmol) was added,the mixture was stirred overnight at room temperature, followed byaddition of 15 ml of DCE. The organic phase was washed with water, anddried over sodium sulfate. The solvent was removed by under reducedpressure to give 200 mg of crude5-bromo-N-(3-(cyclopentyloxy)-4-methoxybenzyl)pyridin-3-amine, which wasused for the next step without further purification.

An Emrys process vial (2-5 ml) for microwave was charged with5-bromo-N-(3-(cyclopentyloxy)-4-methoxybenzyl)pyridin-3-amine (40 mg,0.106 mmol), 4-borono-L-phenylalanine (22 mg, 0.106 mmol) and 2 ml ofacetonitrile. Aqueous sodium carbonate (2 ml, 1M) was added to abovesolution followed by 10 mol percent of dichlorobis(triphenylphosphine)-palladium (II). The reaction vessel was sealed andheated to 180° C. for 10 minutes with a microwave. After cooling, thereaction mixture was evaporated to dryness. The residue was dissolved in2.5 ml of methanol and purified with Prep-LC to give 20 mg of(S)-2-amino-3-(4-(5-3-(cyclophentyloxy-4-methoxy-benzylamino)pyridine-3-yl)phenyl)-propanoicacid. NMR: ¹H-NMR (400 MHz, CD₃OD): δ 1.59 (m, 2H), 1.7 (m, 6H), 3.17(m, 1H), 3.3 (m, 1H), 3.75 (s, 3H), 4.2 (dd, 1H) 4.39 (s, 2H), 4.7 (m,1H), 6.9 (m, 3H), 7.4 (d, 2H), 7.6 (d, 2H), 7.7 (s, 1H), 7.9 (s, 1H),8.15 (s, 1H); Analytical HPLC: RT 2.69; M+1: 462 (RT: 1.285).

6.44. Synthesis of2-Amino-3-(3-(4-amino-6-((R)-1-(naphthalen-2-yl)ethylamino)-1,3,5-triazin-2-yl)phenyl)propanoicacid

To a solution of tert-butyl 2-(diphenylmethylene-amino) acetate (400 mg,1.35 mmol) in THF (25 ml) was added a solution of LDA (1.8M in THF, 2eq, 2.7 mmol, fresh bottle from Aldrich) over 5 minutes at −78° C., andthe resulting mixture was stirred for 20 minutes. A solution of2-(3-(bromomethyl)phenyl)-5,5-dimethyl-1,3,2-dioxaborinane (460 mg, 1.2eq. 1.62 mmol) in THF (10 ml) was added drop-wise to the reactionmixture over 5 minutes. The reaction was continued at same (−78° C.)temperature for 30 minutes, and left for 3 hours at room temperature.The reaction was quenched with saturated NH₄Cl, followed by the additionof water (30 ml), and was extracted with EtOAc (2×40 ml). The organicfractions were combined and dried over Na₂SO₄. The solvent was thenconcentrated at reduced pressure and crudetert-Butyl-3-(3-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl)phenyl)2(diphenylmethylene amino) propionate was purified by columnchromatography to provide the product as a semi-solid.

An Emrys process vial (20 ml) for microwave was charged with(R)-6-chloro-N²-(1-(naphthalene-2-yl)ethyl)-1,3,5-triazine-2,4-diamine(100 mg, 0.33 mmol),tert-butyl-3-(3-(5,5-dimethyl-1,3,2-dioxaborinan-2-yl)phenyl)-2-(diphenylmethyleneamino) propanoate (248 mg, 0.5 mmol, 1.5 eq.) and 6 ml ofacetonitrile plus 6 ml of aqueous sodium carbonate (1M) was added toabove solution followed by 10 mol percent ofdichlorobis(triphenylphosphine)-palladium(II). The reaction vessel wassealed and heated to 190° C. for 10 minutes with microwave. Aftercooling, the reaction mixture was evaporated to dryness. The residue wasdissolved in 10 ml of THF, to which was added 5N.HCl (5 ml). The mixturewas refluxed for 2 hours in order to deprotect the benzophone andtert-butyl groups. The resulting reaction mixture was concentrated anddissolved in methanol (8 ml) and purified with Prep-LC to afford 15 mgof2-amino-3-(4(4-amino-6-((R)-1-(naphthalene-2-yl)ethylamino)-1,3,5-trizin-2-yl)phenyl)propanoicacid. NMR: ¹H-NMR (400 MHz, CD₃OD): δ 1.85 (d, 3H), 3.2-3.45 (m, 2H),4.37 (m, 1H), 5.5 (m, 1H), 7.4 (m, 1H), 7.6 (m 4H), 7.9 (m, 4H), 8.18(m, 2H), Analytical HPLC: RT 2.79 M+1: 429 (RT: 1.35).

6.45. Synthesis of2-Amino-3-(4-(4-amino-6-((R)-1-(naphthalen-2-yl)ethylamino)-1,3,5-triazin-2-yl)-2-fluorophenyl)propanoicacid

To a solution of tert-butyl 2-(diphenylmethylene-amino) acetate (1.1 g,3.73 mmol) in THF (30 ml) was added a solution of LDA (1.8M in THF, 1eq, 3.73 mmol, fresh bottle from Aldrich) over 5 minutes at −78° C., andthe resulting mixture was stirred for 20 minutes. A solution of4-bromo-1-(bromomethyl)-2-fluorobenezene (1 g, 3.74 mmol) in THF (10 ml)was added drop-wise to the reaction mixture over 5 minutes. The reactionwas continued at −78° C. for 30 minutes, after which it was left at roomtemperature for 3 hours. The reaction was quenched with saturated NH₄Cl,after which water (30 ml) was added. Product was extracted with EtOAc(2×40 ml), and the organic fractions were combined and dried overNa₂SO₄. The solvent was concentrated at reduced pressure and crudetert-Butyl3-(4-bromo-2-fluorophenyl)-2-(diphenylmethyleneamino)-propanoate waspurified by column chromatography. The product was obtained as a solid.

An Emrys process vial (20 ml) for microwave was charged with tert-butyl3-(4-bromo-2-fluorophenyl)-2-(diphenylmethylene-amino)propanoate (600mg, 1.24 mmol), Pd(dba)₂ (71 mg, 0.124 mmol), PCy3 (35 mg, 0.124 mmol),4,4,4′,4′,5,5,5′,5′-octamethyl-2,2′-bi(1,3,2-dioxaborolane (346 mg, 1.1eq. 1.36 mmol) and KOAc (182 mg, 1.5 eq., 1.86 mmol) 20 ml of DMF. Thereaction vessel was sealed and heated to 160° C. for 20 minutes bymicrowave. After cooling, the reaction mixture was evaporated to drynessunder reduced pressure. The residue was dissolved in H₂O (30 ml),extracted with EtOAc (2×40 ml), and purified with Prep-LC to give 220 mgof tert-butyl2-(diphenylmethyleneamino)-3-(2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propanoate.

An Emrys process vial (5 ml) for microwave was charged with(R)-6-chloro-N²-(1-(naphthalene-2-yl)ethyl)-1,3,5-triazine-2,4-diamine(67 mg, 0.22 mmol),tert-butyl-2-(diphenylmethyleneamino)-3-(2-fluoro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)propanoate(120 mg, 0.22 mmol) and 2 ml of acetonitrile. Aqueous sodium carbonate(2 ml, 1M) was added to above solution followed by 10 mol percentdichlorobis(triphenylphosphine)-palladium(II). The reaction vessel wassealed and heated to 190° C. for 10 minutes by microwave. After cooling,the reaction mixture was evaporated to dryness. The residue wasdissolved in 10 ml of THF, to which 5N.HCl (2 ml) was then added. Themixture was refluxed for 2 hours (deprotection of benzophone andtert-butyl groups). After deprotection of two groups, the mixture wasconcentrated, dissolved in methanol (5 ml), and purified with Prep-LC toafford 10 mg of2-amino-3-(4-(4-amino-6-((R)-1-(naphthalene-2-yl)ethylamino)-1,3,5-trizin-2-yl)-2-fluorophenyl)propanoicacid. NMR: ¹H-NMR (400 MHz, CD₃OD): δ 1.6 (d, 3H), 3.07 (m, 1H), 3.45(m, 1H), 3.8 (m, 1H), 5.45 (m, 1H), 7.4 (m, 4H), 7.6 (m 1H), 7.8 (m,4H), 8.08 (m, 1H), Analytical HPLC: RT 2.88, M+1: 447 (RT: 1.44).

6.46. Synthesis of(2S)-2-Amino-3-(4-(4-amino-6-(1-(adamantyll)ethylamino)-1,3,5-triazin-2-yl)phenyl)propanoicacid

A solution of adamantine amine (1 equivalent),2-amino-4,6-dichloro-[1,3,5]triazine (1 equivalent) and diisopropylethyl amine (5 equivalents, Aldrich) in anhydrous 1,4-dioxane wasrefluxed at 130° C. for 3 hours. After completion of the reaction, thedioxane was removed under reduced pressure. The reaction was then cooledto room temperature, water was added, and product was extracted withdichloromethane (2×40 ml). The combined organic solution was dried overNa₂SO₄ and concentrated to afford product, which was used in the nextstep without purification.

An Emrys process vial (20 ml) for microwave was charged with adamantinetrizine chloride (200 mg, 0.65 mmol), 4-borono-L-phenylalanine (135 mg,0.65 mmol) and 5 ml of acetonitrile. Aqueous sodium carbonate (5 ml, 1M)was added to above solution followed by 5 mol percentdichlorobis(triphenylphosphine)-palladium(II). The reaction vessel wassealed and heated to 190° C. for 20 minutes by microwave. After cooling,the reaction mixture was evaporated to dryness. The residue wasdissolved in 4 ml of methanol and purified with Prep-LC to give 60 mg(yield 21%) of coupled product. NMR: ¹H-NMR (400 MHz, CD₃OD): δ 1.22 (m,3H), 1.6-1-8 (m, 12H), 2.01 (d, 3H), 3.25-3.42 (m, 2H), 4.0 (m, 1H),4.40 (m, 1H), 7.6 (d, 2H), 8.2 (d, 2H), Analytical HPLC: RT 3.11, M+1:437 (RT: 1.76).

6.47. Alternative Synthesis of(2S)-2-Amino-3-(4-(4-amino-6-(1-(adamantyll)ethylamino)-1,3,5-triazin-2-yl)phenyl)propanoicacid

Adamantane (2-yl)ethyl cyanoguanidine was prepared by forming a solutionof cyanoguanidine (1 equivalent), (S)-2-amino-3-(4-cyanophenylpropanoicacid (1 equivalent) and potassium tertiary butaoxide (3.5 equivalent,Aldrich) in dry n-BuOH, which was vigorously refluxed at 160° C. in asealed tube for 2 days. After completion of the reaction, the mixturewas allowed to cool to room temperature, and the reaction was quenchedwith water. Solvent was removed under reduced pressure. Again, afterallowing to cool to room temperature, the reaction mixture was broughtto pH 12-14 by adding 1N NaOH. Then, impurities were removed whileextracting with Ether:EtOAc (9:1, 2×100 ml). The aqueous solution wascooled to 0° C., 1N HCl was then added to adjust pH to 7. The paleyellow product was slowly crashed out in H₂O, the mixture was kept in arefrigerator for 30 minutes, and the solid was obtained by filtrationwith 92% purity. Compound was crystallized from MeOH to afford a whitesolid (>98% pure, 48-78% yield). ¹H-NMR (400 MHz, CD₃OD): δ 1.0 (d, 3H),1.45-1.6 (m, 6H), 4.62-4.8 (m, 4H) 2.0 (m, 2H), 3.3 (m, 1H), 3.5 (m,1H); Analytical HPLC: RT 2.69; M+1: 462 (RT: 1.285).

The title compound was prepared from adamantane (2-yl)ethylcyanoguanidine using the method shown in Scheme 6.

6.48. Synthesis of(S)-2-Amino-3-(4-(5-fluoro-4-((R)-1-(naphthalen-2-yl)ethylamino)pyrimidin-2-yl)phenyl)propanoicacid

A mixture of (R)-(+)-1-(2-napthyl)ethylamine (102.6 mg, 0.599 mmol),2,4-dichloro-5-fluororo pyrimidine (100 mg, 0.599 mmol) and cesiumcarbonate (390 mg, 1.2 mmol) was dissolved in 1,4-dioxane (3 ml) and H₂O(3 ml) in a 10 ml microwave vial. The mixture was stirred in themicrowave reactor at 80° C. for 10 minutes. The residue was dissolved inCH₂Cl₂ (50 ml), washed with water (20 ml), brine (20 ml) dried (Na₂SO₄)and concentrated to get the crude intermediate2-chloro-5-fluoro-pyrimidin-4-yl)-(1-naphthalen-2-yl-ethyl)-amine.

The crude intermediate (250 mg, 0.83 mmol) was then dissolved in 6.0 mlof MeCN and 6 ml of H₂O in a 20 ml microwave vial. To this solution wereadded L-p-borono-phenylalanine (173.6 mg, 0.83 mmol), sodium carbonate(173.6 mg, 1.66 mmol) and catalytic amount ofdichlorobis(triphenylphosphine)-palladium(I) (11.6 mg, 0.0166 mmol). Thereaction vial was then sealed and stirred in the microwave reactor at150° C. for 7 minutes. The contents were then filtered, and the filtratewas concentrated and dissolved in MeOH and H₂O (1:1) and purified bypreparative HPLC using MeOH/H₂O/TFA as the solvent system. The combinedpure fraction were evaporated in vacuo and further dried on alyophilizer to give 154 mg of2-amino-3-{4-[5-fluoro-4-(1-naphthalen-2-yl-ethylamino)-pryrimidin-2-yl]-phenyl}-propionicacid. NMR: ¹H-NMR (400 MHz, CD₃OD) δ 1.8 (d, 3H) 3.2-3.4 (m, 2H), 4.35(m, 1H), 5.7 (q, 1H), 7.5 (m, 4H), 7.6 (d, 1H), 7.8-7.9 (m, 4H), 8.1 (d,2H), 8.3 (d, 1H). LCMS: M+1=431.

6.49. Synthesis of(S)-2-Amino-3-(4-(2-amino-6-(4-(trifluoromethyl)-benzylamino)pyrimidin-4-yl)phenyl)propanoicacid

A mixture of trifluoromethyl benzylamine (106.8 mg, 0.610 mmol),2-amino-4,6-dichloropyrimidine (100 mg, 0.610 mmol) and cesium carbonate(217 mg, 1.2 mmol) was dissolved in 1,4-dioxane (6 ml) and H₂O (6 ml) ina 20 ml microwave vial. The mixture was stirred in the microwave reactorat 210° C. for 25 minutes. The solvent was then removed. The residue wasdissolved in CH₂Cl₂ (50 ml), washed with water (20 ml), brine (20 ml),dried (Na₂SO₄) and concentrated to get the crude intermediate6-chloro-N-4′-(trifluoromethyl-benzyl)-pryrimidine-2-4-diamine.

The crude intermediate (150 mg, 0.497 mmol) was then dissolved in 3.0 mlof MeCN and 3 ml of H₂O in a 10 ml microwave vial. To this solution wereadded L-p-borono-phenylalanine (104 mg, 0.497 mmol), sodium carbonate(150 mg, 0.994 mmol) and catalytic amount ofdichlorobis(triphenylphosphine)-palladium(II) (6.9 mg, 0.00994 mmol).The reaction vial was then sealed and stirred in the microwave reactorat 150° C. for 5 minutes. The contents were filtered, and the filtratewas concentrated and dissolved in MeOH and H₂O (1:1) and purified bypreparative HPLC using a MeOH/H₂O/TFA solvent system. The combined purefractions were evaporated in vacuo and further dried on a lyophilizer toafford2-amino-3-{4-[2-amino-6-(4-trifluoromethyl-benzylamino)-pyrimidin-4-yl]-phenyl}-propionicacid. NMR: ¹H-NMR (300 MHz, CD₃OD) δ 3.1-3.3 (m, 2H), 4.2 (t, 1H), 4.7(s, 2H), 6.3 (s, 1H), 7.4-7.5 (m, 4H), 7.6 (d, 2H), 7.7 (d, 2H). LCMS:M+1=432.

6.50. Synthesis of2-Amino-3-(5-(5-phenylthiophen-2-yl)-1H-indol-3-yl)propanoic acid

2-Amino-3-(5-bromo-1H-indol-3-yl)-propionic acid (0.020 g, 0.071 mmol)was added to a 5 ml microwave vial, which contained5-phenyl-thiophen-2-boronic acid (0.016 g, 0.078 mmol), Na₂CO₃ (0.015 g,0.142 mmol), acetonitrile (1.5 ml)/water (1.5 ml) anddichlorobis(triphenylphosphine)-palladium (3 mg, 0.003 mmol). Microwavevial was capped and stirred at 150° C. for 5 min under microwaveradiation. Reaction mixture was cooled, filtered through a syringefilter and then separated by a reverse phase preparative-HPLC usingYMC-Pack ODS 100×30 mm ID column (MeOH/H₂O/TFA solvent system). The purefractions were concentrated in vacuum. The product was then suspended in5 ml of water, frozen and lyophilized to give 5 mg of pure product,2-amino-3-[5-(5-phenyl-thiophen-2-yl)-1H-indol-3-yl]-propionic acid.¹H-NMR (300 MHz, CD₃OD): 3.21-3.26 (m, 2H), 4.25 (q, 1H), 7.15-7.35 (m,8H), 7.58 (d, 2H), 7.82 (d, 1H).

6.51. Synthesis of(S)-2-Amino-3-(4-(4-(4-phenoxyphenyl)-1H-1,2,3-triazol-1-yl)phenyl)propanoicacid

A mixture of 1-ethynyl-4-phenoxy-benzene (126 mg, 0.65 mmol) and(S)-3-(4-azido-phenyl)-2-tert-butoxycarbonylamino-propionic acid (200mg, 0.65 mg) in H₂O:dioxane (5:1) was heated at 100° C. in a sealed tubefor overnight. After completion of reaction, 3N HCl (5 ml) was added andthe mixture was stirred for 2 hr at 50° C. Removal of solvent gave crudeproduct which was dissolved in MeOH and purified by preparative HPLC togive 45 mg of desired product (yield: 29%). ¹H-NMR (400 MHz, CD₃OD): δ(ppm) 3.2 (m, 1H), 3.4 (m, 1H), 4.3 (m, 1H), 6.9 (d, 2H), 7.0 (d, 2H),7.2 (m, 1H), 7.3 (d, 2H), 7.4-7.55 (m, 6H), 8.0 (s, 1H).

6.52. Synthesis of(S)-2-Amino-3-(4-(4-(4-(thiophene-2-carboxamido)phenyl)-1H-1,2,3-triazol-1-yl)phenyl)propanoicacid and(S)-2-Amino-3-(4-(5-(4-(thiophene-2-carboxamido)phenyl)-1H-1,2,3-triazol-1-yl)phenyl)propanoicacid

A mixture of thiophene-2-carboxylic acid (4-ethyl-phenyl) amide (117 mg,0.49 mmol) and(S)-3-(4-azido-phenyl)-2-tert-butoxycarbonylamino-propionic acid (150mg, 0.49 mg) in 5 ml of H₂O:dioxane (5:1) was heated at 100° C. in asealed tube overnight. After completion of reaction, 3N HCl (5 ml) wasadded and the mixture was stirred for 2 hr at 50° C. Removal of solventgave crude product which was dissolved in MeOH and purified bypreparative HPLC. According to LCMS (retention time) and NMR, tworegio-isomers were obtained (total yield: 70 mg, 66%). The major productis(S)-2-amino-3-(4-(4-(4-(thiophene-2-carboxamido)phenyl)-1H-1,2,3-triazol-1-yl)phenyl)propanoicacid. NMR: ¹H-NMR (400 MHz, CD₃OD): δ 3.2 (m, 1H), 3.4 (m, 1H), 4.3 (m,1H), 7.15 (m, 1H), 7.3 (d, 2H), 7.6 (m, 4H), 7.0 (m, 3H), 7.95 (d, 1H),8.0 (s, 1H). The minor product is(S)-2-amino-3-(4-(5-(4-(thiophene-2-carboxamido)phenyl)-1H-1,2,3-triazol-1-yl)phenyl)propanoicacid. ¹H-NMR (400 MHz, CD₃OD): δ 3.2 (m, 1H), 3.4 (m, 1H), 4.35 (m, 1H),7.2 (m, 1H), 7.3 (d, 2H), 7.5-7.6 (m, 4H), 7.75 (m, 3H), 7.95 (d, 1H),8.05 (s, 1H).

6.53. Synthesis of(S)-2-Amino-3-(4-(2-amino-6-(phenylethynyl)pyrimidin-4-yl)phenyl)propanoicacid

2-Amino 4,6-dichloro pyrimidine (0.180 g, 1.1 mmol),trimethyl-phenylethynyl-stannane (0.264 g, 1 mmol), were dissolved inTHF (20 ml) and the mixture was stirred at 65° C. for 12 h. LCMSindicated the completion of reaction. Solvent was removed and theresidue was directly used in the following step.

The crude intermediate (0.42 g), L-p-borono-phenylalanine (0.210 g, 1mmol), sodium carbonate (0.210 g, 2 mmol), and dichlorobis(triphenylphosphine)-palladium(II) (25 mg, 0.036 mmol) were dissolved ina mixture of MeCN (3 ml) and H₂O (3 ml) in a 10 ml microwave vial. Thevial was sealed and stirred in the microwave reactor at 150° C. for 6min. The mixture was filtered and the filtrate was concentrated. Residuewas purified by preparative HPLC using MeOH/H₂O/TFA as solvent system toobtain(S)-2-amino-3-[4-(2-amino-6-phenylethynyl-pyrimidin-4-yl(-phenyl]-propionicacid as a TFA salt. ¹H-NMR (400 MHz, CD₃OD): δ (ppm) 3.20-3.42 (m, 2H),4.31 (m, 1H), 7.40-7.51 (m, 6H), 7.62 (d, 2H), 8.18 (d, 2H).

6.54. Additional Compounds

Additional compounds prepared using methods known in the art and/ordescribed herein are listed below:

LCMS HPLC Method Compound (M + 1) (Time (min))(S)-2-amino-3-(4-(5-(2-fluoro-4,5- 426 C (3.04)dimethoxybenzylamino)pyrazin-2-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(2-amino-6-(4-(2-methoxyphenyl)piperidin-1- 448 I(3.03) yl)pyrimidin-4-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(6-(3-(cyclopentyloxy)-4- 507 J (3.21)methoxybenzylamino)-2-(dimethylamino)pyrimidin-4- yl)phenyl)propanoicacid (S)-2-amino-3-(4-(5-(3,4-dimethylbenzylamino)pyrazin-2- 377 C(3.15) yl)phenyl)propanoic acid(S)-2-amino-3-(4-(5-(biphenyl-2-ylmethylamino)pyrazin-2- 425 D (4.00)yl)phenyl)propanoic acid (S)-ethyl 2-amino-3-(4-(2-amino-6-(4- 460 F(2.52) (trifluoromethyl)benzylamino)pyrimidin-4-yl)phenyl)propanoate(S)-2-amino-3-(4-(5-(cyclopentylmethylamino)pyrazin-2- 341 C (2.77)yl)phenyl)propanoic acid (2S)-2-amino-3-(4-(2-amino-6-(3-(2- 472 A(2.87) (trifluoromethyl)phenyl)pyrrolidin-1-yl)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1,2,3,4-tetrahydronaphthalen-1- 404 A(2.65) ylamino)pyrimidin-4-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(2-amino-6-((R)-1-(naphthalen-2- 429 A (2.73)yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1,2- 454 K (1.34)diphenylethylamino)pyrimidin-4-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(2-amino-6-((R)-1-(4-(benzo[b]thiophen-3- 510 D (2.02)yl)phenyl)ethylamino)pyrimidin-4-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(4-amino-6-((R)-1-(4′-methoxybiphenyl-4- 485 J (2.99)yl)ethylamino)-1,3,5-triazin-2-yl)phenyl)propanoic acid2-amino-3-(1-(4-amino-6-((R)-1-(naphthalen-2-yl)ethylamino)- 436 B(2.25) 1,3,5-triazin-2-yl)piperidin-4-yl)propanoic acid(2S)-2-amino-3-(4-(4-amino-6-(1-(4-fluoronaphthalen-1- 447 H (1.68)yl)ethylamino)-1,3,5-triazin-2-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(4-amino-6-((3′-fluorobiphenyl-4- 459 J (2.89)yl)methylamino)-1,3,5-triazin-2-yl)phenyl)propanoic acid2-amino-3-(4-(4-amino-6-((R)-1-(naphthalen-2-yl)ethylamino)- 447 A(2.88) 1,3,5-triazin-2-yl)-2-fluorophenyl)propanoic acid(S)-2-amino-3-(4-(2-amino-6-((R)-2,2,2-trifluoro-1-(3′- 539 M (3.83)methoxybiphenyl-4-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(4-amino-6-(2,2,2-trifluoro-1-(3′- 528 F (3.41)fluorobiphenyl-2-yl)ethoxy)-1,3,5-triazin-2-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(4-amino-6-(1-(4-tert- 435 J (1.82)butylphenyl)ethylamino)-1,3,5-triazin-2-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3′- 527 D (2.09)fluorobiphenyl-4-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(4-amino-6-(6,7-dihydroxy-1-methyl-3,4- 437 B (2.47)dihydroisoquinolin-2(1H)-yl)-1,3,5-triazin-2- yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(4-amino-6-(2,2,2-trifluoro-1-(3′- 524 D (2.22)methylbiphenyl-4-yl)ethoxy)-1,3,5-triazin-2- yl)phenyl)propanoic acid(S)-2-amino-3-(4-(4-amino-6-((R)-1-(naphthalen-2- 428 A (2.90)yl)ethylamino)pyrimidin-2-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(2-amino-6-(benzylthio)pyrimidin-4- 379 E (1.66)yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(4′- 527 E (2.07)fluorobiphenyl-4-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(6-(3-(4-chlorophenoxy)piperidin-1- 453 A (2.67)yl)pyrimidin-4-yl)phenyl)propanoic acid(S)-3-(4-(4-amino-6-((R)-1-(naphthalen-2-yl)ethylamino)-1,3,5- 486 J(2.83) triazin-2-yl)phenyl)-2-(2-aminoacetamido)propanoic acid(S)-2-amino-3-(4-(6-((R)-1-(naphthalen-2-yl)ethylamino)-2- 481 A (3.70)(trifluoromethyl)pyrimidin-4-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(2-amino-6-(4-(3-chlorophenyl)piperazin-1- 453 L (0.72)yl)pyrimidin-4-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(2-amino-6-((R)-2,2,2-trifluoro-1- 433 E (1.77)phenylethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1,4- 482 A (3.15)diphenylbutylamino)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(6-(1-(3′-chlorobiphenyl-2-yl)-2,2,2- 528 E (2.35)trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(4-amino-6-(1-(biphenyl-4-yl)-2,2,2- 510 D (2.14)trifluoroethoxy)-1,3,5-triazin-2-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,3,3,3-pentafluoro-1-(3- 515 N (3.34)fluoro-4-methylphenyl)propoxy)pyrimidin-4- yl)phenyl)propanoic acid(S)-ethyl 2-amino-3-(4-(2-amino-6-((R)-2,2,2-trifluoro-1-(3′- 567 N(2.17) methoxybiphenyl-4-yl)ethoxy)pyrimidin-4- yl)phenyl)propanoate(S)-2-amino-3-(4-(2-amino-6-((S)-2,2,2-trifluoro-1-(3′- 539 N (3.36)methoxybiphenyl-4-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3-fluoro-3′- 557 O(3.52) methoxybiphenyl-4-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(3′-(dimethylamino)biphenyl- 552 Q(3.00) 2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3′-methoxy-5- 553 N(3.63) methylbiphenyl-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(4′-methoxy-5- 553 N(3.61) methylbiphenyl-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3′-methoxy-3- 617 O(3.28) (methylsulfonyl)biphenyl-4-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(2-(cyclopropylmethoxy)-4- 521 N (1.57)fluorophenyl)-2,2,2-trifluoroethoxy)pyrimidin-4- yl)phenyl)propanoicacid (2S)-2-amino-3-(4-(6-(1-(2-(cyclopropylmethoxy)-4- 507 N (1.62)fluorophenyl)-2,2,2-trifluoroethoxy)pyrimidin-4- yl)phenyl)propanoicacid (2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(2- 520 N (1.69)(isopentyloxy)phenyl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(5-(2,2,2-trifluoro-1-(3′-fluorobiphenyl-4- 512 —yl)ethoxy)pyrazin-2-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(4′- 539 N (3.50)methoxybiphenyl-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(3′-carbamoylbiphenyl-2-yl)- 552 N(3.14) 2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(4′-carbamoylbiphenyl-2-yl)- 552 N(3.05) 2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(4-(2- 555 N (1.55)methoxyphenoxy)phenyl)ethoxy)pyrimidin-4- yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(6-(2,2,2-trifluoro-1-(4-(2- 541 N (1.59)methoxyphenoxy)phenyl)ethoxy)pyrimidin-4- yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(6-(2,2,2-trifluoro-1-(2- 505 N (1.74)(isopentyloxy)phenyl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-3-(4-(6-(1-(3′-acetamidobiphenyl-2-yl)-2,2,2- 566 N (3.18)trifluoroethoxy)-2-aminopyrimidin-4-yl)phenyl)-2- aminopropanoic acid(2S)-3-(4-(6-(1-(4′-acetamidobiphenyl-2-yl)-2,2,2- 566 N (3.23)trifluoroethoxy)-2-aminopyrimidin-4-yl)phenyl)-2- aminopropanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(4-cyanophenyl)-2,2,2- 458 —trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid (S)-ethyl2-amino-3-(4-(2-amino-6-((R)-2,2,2-trifluoro-1-p- 475 —tolylethoxy)pyrimidin-4-yl)phenyl)propanoate(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(1- 493 O (2.97)methoxybicyclo[2.2.2]oct-5-en-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(4-(cyclopentyloxy)phenyl)- 517 N (1.61)2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(6-(1-(4-(cyclopentyloxy)phenyl)-2,2,2- 503 N (1.67)trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(4-(3- 556 N (1.59)methoxyphenoxy)phenyl)ethoxy)pyrimidin-4- yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(4,5-dimethoxybiphenyl-2-yl)- 569 S(3.34) 2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(4,5-dimethoxy-3′- 583 S (3.50)methylbiphenyl-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(5-(2,2,2-trifluoro-1-(2′-methylbiphenyl-2- 508 —yl)ethoxy)pyrazin-2-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(6-(2,2,2-trifluoro-1-(4-(3- 541 N (1.64)methoxyphenoxy)phenyl)ethoxy)pyrimidin-4- yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(2-(3,5- 561 N (1.64)difluorophenoxy)phenyl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(4-(4- 556 N (1.58)methoxyphenoxy)phenyl)ethoxy)pyrimidin-4- yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(4′-((S)-2-amino-2- 596 —carboxyethyl)biphenyl-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(2-bromophenyl)-2,2,2- 513 —trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(5-(2,2,2-trifluoro-1-(3′-methylbiphenyl-2- 508 —yl)ethoxy)pyrazin-2-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(4- 539 S (3.51)methoxybiphenyl-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(5-(2,2,2-trifluoro-1-(2-(4-methylthiophen-3- 514 —yl)phenyl)ethoxy)pyrazin-2-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(4-methoxy-3′- 553 S(3.66) methylbiphenyl-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3′- 539 —(hydroxymethyl)biphenyl-2-yl)ethoxy)pyrimidin-4- yl)phenyl)propanoicacid (2S)-2-amino-3-(4-(2-amino-6-(1-(3′-cyanobiphenyl-2-yl)-2,2,2- 534— trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(6-(1-(2-(3,5-difluorophenoxy)phenyl)-2,2,2- 547 N(1.69) trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(6-(2,2,2-trifluoro-1-(4-(4- 541 N (1.63)methoxyphenoxy)phenyl)ethoxy)pyrimidin-4- yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(2-(4- 536 —methylthiazol-2-yl)thiophen-3-yl)ethoxy)pyrimidin-4- yl)phenyl)propanoicacid (2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(5-(4- 530 O (3.14)methoxyphenyl)isoxazol-3-yl)ethoxy)pyrimidin-4- yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(1-phenyl-5- 567 O(3.24) (trifluoromethyl)-1H-pyrazol-4-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(2-(cyclohexyloxy)-4- 545 N (1.76)methylphenyl)-2,2,2-trifluoroethoxy)pyrimidin-4- yl)phenyl)propanoicacid (2S)-2-amino-3-(4-(2-amino-6-(1-(2-(cyclopentyloxy)-4- 532 N (1.71)methylphenyl)-2,2,2-trifluoroethoxy)pyrimidin-4- yl)phenyl)propanoicacid (2S)-2-amino-3-(4-(2-amino-6-(1-(benzo[d]thiazol-6-yl)-2,2,2- 490 O(2.66) trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(1-methyl-1H- 437 —imidazol-5-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(6-(1-(2-(cyclopentyloxy)-4-methylphenyl)- 517 N(1.78) 2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(6-(1-(2-(cyclohexyloxy)-4-methylphenyl)- 531 N (1.87)2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(pyridin-3- 434 —yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(1,3-dimethyl-1H-pyrazol-5- 451 —yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(2-amino-6-(3-hydroxyphenyl)pyrimidin-4- 351 —yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3′- 526 —hydroxybiphenyl-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(2-amino-6-(3,5-difluorophenyl)pyrimidin-4- 371 —yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(3′,5′-difluorobiphenyl-2-yl)- 546 —2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(6-(2,2,2-trifluoro-1-(3′-fluorobiphenyl-3- 512 —yl)ethoxy)pyrazin-2-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(5-ethoxy-2-methyl-2,3- 533 O (3.16)dihydrobenzofuran-6-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(benzofuran-5-yl)-2,2,2- 473 —trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(2-m- 513 —tolylfuran-3-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid (S)-ethyl3-(4-(2-amino-6-((R)-2,2,2-trifluoro-1-(3′- 596 N (3.55)methoxybiphenyl-4-yl)ethoxy)pyrimidin-4-yl)phenyl)-2-(2-aminoacetamido)propanoate(2S)-2-amino-3-(4-(6-(2,2,2-trifluoro-1-(2-(4-methylthiophen-3- 514 —yl)phenyl)ethoxy)pyrazin-2-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(5-methyl-3- 514 N(3.12) phenylisoxazol-4-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(2-amino-6-(3-(methylthio)phenyl)pyrimidin- 381 —4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3′- 555 —(methylthio)biphenyl-2-yl)ethoxy)pyrimidin-4- yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(3′- 566 —((dimethylamino)methyl)biphenyl-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(2-amino-6-(3- 419 —(trifluoromethoxy)phenyl)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3′- 593 —(trifluoromethoxy)biphenyl-2-yl)ethoxy)pyrimidin-4- yl)phenyl)propanoicacid (S)-3-(4-(2-amino-6-((R)-2,2,2-trifluoro-1-(3′-methoxybiphenyl- 596N (1.51) 4-yl)ethoxy)pyrimidin-4-yl)phenyl)-2-(2-aminoacetamido)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(1-methyl-5- 513 N(2.88) phenyl-1H-pyrazol-4-yl)ethoxy)pyrimidin-4- yl)phenyl)propanoicacid (2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(4- 511 —(methylsulfonyl)phenyl)ethoxy)pyrimidin-4- yl)phenyl)propanoic acid(S)-2-amino-3-(4-(2-amino-6-((R)-1-(3′- 552 S (3.09)(dimethylamino)biphenyl-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid (2S)-2-amino-3-(4-(2-amino-6-(1-(2-chloro-4-545 — (methylsulfonyl)phenyl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(3-(furan-2- 505 —yl)thiophen-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(2-(cyclopentyloxy)-4- 543 N (1.66)fluorophenyl)-2,2,2-trifluoroethoxy)pyrimidin-4- yl)phenyl)propanoicacid (2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(2-(3- 543 O (3.59)methoxyphenyl)cyclohex-1-enyl)ethoxy)pyrimidin-4- yl)phenyl)propanoicacid (2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(pyrimidin-5- 435 —yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(5-(2,2,2-trifluoro-1-(3′-methoxybiphenyl-3- 524 —yl)ethoxy)pyrazin-2-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(2-amino-6-((S)-1-(3′- 552 N (3.08)(dimethylamino)biphenyl-2-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(2-(furan-2- 542 N(2.61) carboxamido)phenyl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(4-chloro-2- 545 —(methylsulfonyl)phenyl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid (S)-isopropyl2-amino-3-(4-(2-amino-6-((R)-2,2,2-trifluoro-1- 581 —(3′-methoxybiphenyl-4-yl)ethoxy)pyrimidin-4- yl)phenyl)propanoate(2S)-2-amino-3-(4-(6-(1-(2-(cyclopentyloxy)-4-fluorophenyl)- 520 N(1.73) 2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(6-(1-(2-(cyclohexyloxy)-4-fluorophenyl)- 534 N (1.81)2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(1-(thiophen-2- 521 O(3.36) yl)cyclohexyl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-(2,2,2-trifluoro-1-(3′-methoxybiphenyl-4- 529 Q(2.30) yl)ethoxy)thiazol-5-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(2-(cyclohexyloxy)-4- 549 N (1.70)fluorophenyl)-2,2,2-trifluoroethoxy)pyrimidin-4- yl)phenyl)propanoicacid (2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(1-(4- 545 O (3.41)methoxyphenyl)cyclohexyl)ethoxy)pyrimidin-4- yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(6-(2,2,2-trifluoro-1-(4-fluoro-2- 450 N (1.50)methylphenyl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(4-fluoro-2- 465 N(1.45) methylphenyl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(oxazol-2- 432 O (1.76)yl(phenyl)methoxy)pyrimidin-4-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(2-amino-6-(1-cyclohexyl-2,2,2- 452 O (3.47)trifluoroethylideneaminooxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(2-(3- 543 N (3.02)(dimethylamino)phenyl)furan-3-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(5- 515 N (3.39)phenylthiophen-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(S)-phenyl 2-amino-3-(4-(2-amino-6-((R)-2,2,2-trifluoro-1-(3′- 615 Q(3.00) methoxybiphenyl-4-yl)ethoxy)pyrimidin-4- yl)phenyl)propanoate(S)-2-amino-3-(4-(2-amino-6-((R)-1-(3′- 566 N (2.60)((dimethylamino)methyl)biphenyl-4-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(1-(3-methoxybenzoyl)-1H-pyrazol-4- 366 O (2.55)yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(6-(2,2,2-trifluoro-1-(5-phenylfuran-2- 484 N (3.65)yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(4-chloro-2-fluorophenyl)- 486 N (3.14)2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(S,E)-2-amino-3-(4-(2-amino-6-(4- 429 N (2.94)(trifluoromethyl)styryl)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(3,4-dichlorophenyl)-2,2,2- 502 N (3.31)trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-(4-chloro-3-fluorophenyl)- 486 N (3.13)2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(2-amino-6-((R)-1-(3′- 552 N (2.66)(dimethylamino)biphenyl-4-yl)-2,2,2-trifluoroethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1-chloro-2,2,2-trifluoro-1-(4- 573 N(3.77) methoxybiphenyl-2-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(6-(2,2,2-trifluoro-1-(5-phenylthiophen-2- 500 N(3.75) yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(5-(4-phenoxyphenyl)-1H-1,2,3-triazol-1- 401 O (3.20)yl)phenyl)propanoic acid (S,E)-2-amino-3-(4-(2-amino-6-(2-(biphenyl-4-437 N (3.17) yl)vinyl)pyrimidin-4-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(4-amino-6-((R)-2,2,2-trifluoro-1-(3′- 539 —methoxybiphenyl-4-yl)ethoxy)pyrimidin-2-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(4′-methoxybiphenyl-4- 428 N (2.78)ylsulfonamido)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(6-(3- 540 N (3.09)methoxyphenyl)pyridin-3-yl)ethoxy)pyrimidin-4- yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(6-(2-fluoro-3- 558 N(3.00) methoxyphenyl)pyridin-3-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid2-amino-3-(5-(4′-methylbiphenyl-4-yl)-1H-indol-3-yl)propanoic 371 N(1.48) acid 2-amino-3-(5-m-tolyl-1H-indol-3-yl)propanoic acid 295 N(1.19) (2S)-2-amino-3-(4-(2-(2-methoxyphenyl)furan-3- 358 O (2.68)carboxamido)phenyl)propanoic acid2-amino-3-(5-(1-benzyl-1H-pyrazol-4-yl)-1H-indol-3- 361 N (1.10)yl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(2,2,2-trifluoro-1-(6-(thiophen-2- 516 N(1.42) yl)pyridin-3-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoic acid2-amino-3-(6-(1-benzyl-1H-pyrazol-4-yl)-1H-indol-3- 361 N (1.09)yl)propanoic acid(S)-2-amino-3-(4-((2-(4-(trifluoromethyl)phenyl)thiazol-4- 422 O (3.00)yl)methylamino)phenyl)propanoic acid(S)-2-amino-3-(4-((4′-methoxybiphenyl-4- 441 O (2.94)ylsulfonamido)methyl)phenyl)propanoic acid(S)-2-amino-3-(4-(3-(2-methoxydibenzo[b,d]furan-3- 420 O (3.36)yl)ureido)phenyl)propanoic acid (S)-2-amino-3-(4-(3-(2,2- 404 O (2.97)diphenylethyl)ureido)phenyl)propanoic acid(S)-2-amino-3-(4-(phenylethynyl)phenyl)propanoic acid 266 N (2.91)(S)-2-amino-3-(4-(2-amino-6-((5-(1-methyl-5-(trifluoromethyl)- 410 N(1.39) 1H-pyrazol-3-yl)thiophen-2-yl)methoxy)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(1,1,1-trifluoro-3-((R)-2,2,3- 479 O (3.42)trimethylcyclopent-3-enyl)propan-2-yloxy)pyrimidin-4-yl)phenyl)propanoic acid (2S)-2-amino-3-(4-(2-amino-6-(3-(2- 429 N(1.53) hydroxyethylcarbamoyl)piperidin-1-yl)pyrimidin-4-yl)phenyl)propanoic acid(2S)-2-amino-3-(4-(2-amino-6-(3-(pyridin-2-yloxy)piperidin-1- 435 N(2.11) yl)pyrimidin-4-yl)phenyl)propanoic acid(S)-2-amino-3-(4-(2-amino-6-(4-chloro-3-(piperidine-1- 480 N (2.75)carbonyl)phenyl)pyrimidin-4-yl)phenyl)propanoic acid

6.55. In Vitro Inhibition Assays

Human TPH1, TPH2, tyrosine hydroxylase (TH) and phenylalaninehydroxylase (PH) were all generated using genes having the followingaccession numbers, respectively: X52836, AY098914, X05290, and U49897.

The full-length coding sequence of human TPH1 was cloned into thebacterial expression vector pET24 (Novagen, Madison, Wis., USA). Asingle colony of BL21(DE3) cells harboring the expression vector wasinoculated into 50 ml of L broth (LB)-kanamycin media and grown up at37° C. overnight with shaking. Half of the culture (25 ml) was thentransferred into 3 L of media containing 1.5% yeast extract, 2% BactoPeptone, 0.1 mM tryptophan, 0.1 mM ferrous ammonium sulfate, and 50 mMphosphate buffer (pH 7.0), and grown to OD₆₀₀=6 at 37° C. with oxygensupplemented at 40%, pH maintained at 7.0, and glucose added. Expressionof TPH1 was induced with 15% D-lactose over a period of 10 hours at 25°C. The cells were spun down and washed once with phosphate bufferedsaline (PBS).

TPH1 was purified by affinity chromatography based on its binding topterin. The cell pellet was resuspended in a lysis buffer (100 ml/20 g)containing 50 mM Tris-Cl, pH 7.6, 0.5 M NaCl, 0.1% Tween-20, 2 mM EDTA,5 mM DTT, protease inhibitor mixture (Roche Applied Science,Indianapolis, Ind., USA) and 1 mM phenylmethanesulfonyl fluoride (PMSF),and the cells were lyzed with a microfluidizer. The lysate wascentrifuged and the supernatant was loaded onto a pterin-coupledsepharose 4B column that was equilibrated with a buffer containing 50 mMTris, pH 8.0, 2 M NaCl, 0.1% Tween-20, 0.5 mM EDTA, and 2 mM DTT. Thecolumn was washed with 50 ml of this buffer and TPH1 was eluded with abuffer containing 30 mM NaHCO₃, pH 10.5, 0.5 M NaCl, 0.1% Tween-20, 0.5mM EDTA, 2 mM DTT, and 10% glycerol. Eluted enzyme was immediatelyneutralized with 200 mM KH₂PO₄, pH 7.0, 0.5 M NaCl, 20 mM DTT, 0.5 mMEDTA, and 10% glycerol, and stored at −80° C.

Human tryptophan hydroxylase type II (TPH2), tyrosine hydroxylase (TH)and phenylalanine hydroxylase (PAH) were expressed and purifiedessentially in the same way, except the cells were supplemented withtyrosine for TH and phenylalanine for PAH during growth.

TPH1 and TPH2 activities were measured in a reaction mixture containing50 mM 4-morpholinepropanesulfonic acid (MOPS), pH 7.0, 60 μM tryptophan,100 mM ammonium sulfate, 100 μM ferrous ammonium sulfate, 0.5 mMtris(2-carboxyethyl)phosphine (TCEP), 0.3 mM 6-methyl tetrahydropterin,0.05 mg/ml catalase, and 0.9 mM DTT. The reactions were initiated byadding TPH1 to a final concentration of 7.5 nM. Initial velocity of thereactions was determined by following the change of fluorescence at 360nm (excitation wavelength=300 nm). TPH1 and TPH2 inhibition wasdetermined by measuring their activities at various compoundconcentrations, and the potency of a given compound was calculated usingthe equation:

$v = {b + \frac{v_{0} - b}{1 + \left( \frac{\lbrack C\rbrack}{\left\lbrack I_{c\; 50} \right\rbrack} \right)^{D}}}$where v is the initial velocity at a given compound concentration C, v ois the v when C=0, b is the background signal, D is the Hill slope whichis approximately equal to 1, and I_(c50) is the concentration of thecompound that inhibits half of the maximum enzyme activity.

Human TH and PAH activities were determined by measuring the amount of³H₂O generated using L-[3,4-³H]-tyrosine and L-[4-³H]-phenylalanine,respectively. The enzyme (100 nM) was first incubated with its substrateat 0.1 mM for about 10 minutes, and added to a reaction mixturecontaining 50 mM MOPS, pH 7.2, 100 mM ammonium sulfate, 0.05% Tween-20,1.5 mM TCEP, 100 μM ferrous ammonium sulfate, 0.1 mM tyrosine orphenylalanine, 0.2 mM 6-methyl tetrahydropterin, 0.05 mg/ml of catalase,and 2 mM DTT. The reactions were allowed to proceed for 10-15 minutesand stopped by the addition of 2 M HCl. The mixtures were then filteredthrough activated charcoal and the radioactivity in the filtrate wasdetermined by scintillation counting. Activities of compounds on TH andPAH were determined using this assay and calculated in the same way ason TPH1 and TPH2.

6.56. Cell-Based Inhibition Assays

Two types of cell lines were used for screening: RBL2H3 is a ratmastocytoma cell line, which contains TPH1 and makes5-hydroxytrypotamine (5HT) spontaneously; BON is a human carcinoid cellline, which contains TPH1 and makes 5-hydroxytryptophan (5HTP). The CBAswere performed in 96-well plate format. The mobile phase used in HPLCcontained 97% of 100 mM sodium acetate, pH 3.5 and 3% acetonitrile. AWaters C18 column (4.6×50 mm) was used with Waters HPLC (model 2795). Amulti-channel fluorometer (model 2475) was used to monitor the flowthrough by setting at 280 nm as the excitation wavelength and 360 nm asthe emission wavelength.

RBL CBA: Cells were grown in complete media (containing 5% bovine serum)for 3-4 hours to allow cells to attach to plate wells (7K cell/well).Compounds were then added to each well in the concentration range of0.016 μM to 11.36 μM. The controls were cells in complete media withoutany compound present. Cells were harvested after 3 days of incubation at37° C. Cells were >95% confluent without compound present. Media wereremoved from plate and cells were lysed with equal volume of 0.1 N NaOH.A large portion of the cell lysate was treated by mixing with equalvolume of 1M TCA and then filtered through glass fiber. The filtrateswere loaded on reverse phase HPLC for analyzing 5HT concentrations. Asmall portion of the cell lysate was also taken to measure proteinconcentration of the cells that reflects the cytotoxicity of thecompounds at the concentration used. The protein concentration wasmeasured by using BCA method.

The average of 5HT level in cells without compound treated was used asthe maximum value in the IC₅₀ derivation according to the equationprovided above. The minimum value of 5HT is either set at 0 or fromcells that treated with the highest concentration of compound If acompound is not cytotoxic at that concentration.

BON CBA: Cells were grown in equal volume of DMEM and F12K with 5%bovine serum for 3-4 hours (20K cell/well) and compound was added at aconcentration range of 0.07 μM to 50 μM. The cells were incubated at 37°C. overnight. Fifty μM of the culture supernatant was then taken for5HTP measurement. The supernatant was mixed with equal volume of 1M TCA,then filtered through glass fiber. The filtrate was loaded on reversephase HPLC for 5HTP concentration measurement. The cell viability wasmeasured by treating the remaining cells with Promega Celltiter-GloLuminescent Cell Viability Assay. The compound potency was thencalculated in the same way as in the RBL CBA.

6.57. In Vivo Effects

The in vivo effects of a potent TPH1 inhibitor of the invention wereevaluated in several studies by determining the change of 5-HT levels inthe intestines and brains of mice following oral administration of thecompound.

The compound was formulated in different vehicles to provide either asuspension or solution. Generally, 14-week-old male C57 albino mice weredosed once daily by oral gavage at 5 ml/kg for four consecutive days.Five hours after the last dose, the animals were quickly sacrificed.Various regions of the intestinal tract and whole brain were taken andfrozen immediately. 5-HT was extracted from the tissues and measured byHPLC. Blood samples were taken for exposure analysis.

The potent TPH1 inhibitor was found to reduce 5-HT levels in both thesmall and large intestine, but not in the brain. In one study, thecompound was formulated in H₂O and administered to mice at fourdifferent dose levels: 15, 50, 150, and 500 mg/kg, once daily by oralgavage. As shown in FIG. 1, the compound caused significant reduction of5-HT in the jejunum and ileum in a dose-dependent fashion. In the colon,statistically significant reduction of 5-HT was seen at the 50, 150, and500 mg/kg/day dose levels. No significant change of 5-HT levels wasobserved in the brain at any of the dose levels.

All publications (e.g., patents and patent applications) cited above areincorporated herein by reference in their entireties.

1. A method of treating pulmonary hypertension, which comprisesadministering to a patient in need thereof therapeutically effectiveamounts of an endothelin receptor antagonist and a compound of theformula:

or a pharmaceutically acceptable salt thereof, wherein: A is optionallysubstituted cycloalkyl, aryl, or heterocycle; X is a bond, —O—, —S—,—C(O)—, —C(R₄)═, ═C(R₄)—, —C(R₃R₄)—, —C(R₄)═C(R₄)—, —C≡C—, —N(R₅)—,—N(R₅)C(O)N(R₅)—, —C(R₃R₄)N(R₅)—, —N(R₅)C(R₃R₄)—, —ONC(R₃)—, —C(R₃)NO—,—C(R₃R₄)O—, —OC(R₃R₄)—, —S(O₂)—, —S(O₂)N(R₅)—, —N(R₅)S(O₂),—C(R₃R₄)S(O₂)—, or —S(O₂)C(R₃R₄)—; D is optionally substituted aryl orheterocycle; E is optionally substituted aryl or heterocycle; R₁ ishydrogen or optionally substituted alkyl, alkyl-aryl, alkyl-heterocycle,aryl, or heterocycle; R₂ is hydrogen or optionally substituted alkyl,alkyl-aryl, alkyl-heterocycle, aryl, or heterocycle; R₃ is hydrogen,alkoxy, amino, cyano, halogen, hydroxyl, or optionally substitutedalkyl; R₄ is hydrogen, alkoxy, amino, cyano, halogen, hydroxyl, oroptionally substituted alkyl or aryl; R₅ is hydrogen or optionallysubstituted alkyl or aryl; and n is 0-3.
 2. The method of claim 1,wherein the compound is of the formula:

wherein: each of Z″₁, Z″₂, Z″₃, and Z″₄ is independently N or CR₁₀; eachR₁₀ is independently amino, cyano, halogen, hydrogen, OR₁₁, SR₁₁,NR₁₂R₁₃, or optionally substituted alkyl, alkyl-aryl oralkyl-heterocycle; each R₁₁ is independently hydrogen or optionallysubstituted alkyl, alkyl-aryl or alkyl-heterocycle; each R₁₂ isindependently hydrogen or optionally substituted alkyl, alkyl-aryl oralkyl-heterocycle; and each R₁₃ is independently hydrogen or optionallysubstituted alkyl, alkyl-aryl or alkyl-heterocycle.
 3. The method ofclaim 2, wherein the compound is of the formula:


4. The method of claim 3, wherein A is optionally substituted biphenyl.5. The method of claim 3, wherein R₃ is trifluoromethyl.
 6. The methodof claim 3, wherein E is optionally substituted phenyl.
 7. The method ofclaim 3, wherein R₁₀ is amino.
 8. The method of claim 1, wherein theendothelin receptor antagonist is ambrisentan, BMS-193884, bosentan,darusentan, SB-234551, sitaxsentan, or tezosentan.
 9. The method ofclaim 8, wherein the endothelin receptor antagonist is bosentan.
 10. Amethod of treating pulmonary hypertension, which comprises administeringto a patient in need thereof therapeutically effective amounts of anendothelin receptor antagonist and a compound of the formula:

or a pharmaceutically acceptable salt thereof, wherein A is optionallysubstituted cycloalkyl, aryl, or heterocycle; and R₂ is hydrogen orlower alkyl.
 11. The method of claim 10, wherein A is optionallysubstituted biphenyl.
 12. The method of claim 10, wherein the endothelinreceptor antagonist is ambrisentan, BMS-193884, bosentan, darusentan,SB-234551, sitaxsentan, or tezosentan.
 13. The method of claim 12,wherein the endothelin receptor antagonist is bosentan.
 14. A method oftreating pulmonary hypertension, which comprises administering to apatient in need thereof therapeutically effective amounts of anendothelin receptor antagonist and(S)-2-amino-3-(4-(2-amino-6-((R)-2,2,2-trifluoro-1-(3′-methoxybiphenyl-4-yl)ethoxy)pyrimidin-4-yl)phenyl)propanoicacid, or a pharmaceutically acceptable salt thereof.
 15. The method ofclaim 14, wherein the endothelin receptor antagonist is ambrisentan,BMS-193884, bosentan, darusentan, SB-234551, sitaxsentan, or tezosentan.16. The method of claim 15, wherein the endothelin receptor antagonistis bosentan.